Although scientists generally agree that species represent the most basic and fundamental groups into which living and extinct organisms are classified, there is debate over how species should be identified as well as the precise definition of the word "species." This controversy, known as the species problem, stems from the quest for a single, universal, and functional conception of species. This "essential species concept" would have to identify species unambiguously as discreet, natural units in addition to explaining the existence of species and how they arise in nature. While a number of different species concepts have been proposed, none are able to meet these requirements for all species.

In response to this problem, many scientists recommend a pluralistic approach, that is, the recognition that there are multiple ways to think about and define species. Investigators must therefore choose an appropriate species concept based on their needs. For example, a paleontologist who tries to understand prehistoric vertebrates by studying fossils is likely to require and apply a different conceptualization of species than a microbiologist who studies heredity and variation in populations of bacteria.

Scientists working to understand evolution and speciation (the processes by which species arise), frequently apply the biological species concept. Ernst Mayr, one of the leading evolutionary biologists of the 20th century, provided the most commonly accepted version of the biological species concept, which posits that "species are groups of interbreeding natural populations that are reproductively isolated from other such groups."

Mayer's work focused on vertebrate animals, a group for which the biological species concept is particularly useful. It also has been applied to plants and other populations. However, most plant biologists also consider evolutionary history and morphology in their species definitions. The biological species concept defines species in terms of interbreeding populations, and is therefore most useful for understanding organisms that reproduce sexually. For these organisms, the formation of a new species involves the accumulation of enough genetic differences within individuals of a population to prevent them from breeding with individuals of other populations - a condition referred to as reproductive isolation. When reproductively isolated, species accumulate genetic changes and evolve along independent trajectories. Without reproductive isolation, populations merge in a genetic sense (they share a common gene pool), and thus converge (become similar) in an evolutionary sense.

Many biologists do not interpret the biological species concept rigidly, and characterize species as populations with “substantial but not necessarily complete reproductive isolation” (Coyne and Orr, 2004).

The divergence of species from parental or ancestral populations is reinforced by reproductive barriers that limit exchange of genes with members of other populations. These isolating barriers, originally described by Dobzhanky (1937) as isolation mechanisms, are defined as biological properties of individual organisms that impede gene exchange with members of other populations. External factors, such as geographic barriers are not considered to be reproductive isolating barriers.

Reproductive isolation barriers can be categorized in different ways: relative to the act of mating or relative to the production of zygotes. Premating/prezygotic isolation barriers impede mating between members of different species. However, if members of different species do mate, postmating/prezygotic isolation barriers prevent the formation of a zygote. If these isolating barriers fail and a zygote is formed after the members of two species mate, postmating/postzygotic isolation barriers reduce the viability and/or fertility of the zygote.

Ecological isolation occurs when different species live in the same geographic area but occupy different habitats within that area. These barriers are byproducts of different adaptations to local environments. Under these circumstances, individuals of different species do not hybridize simply because they rarely encounter one another.

For example, until recently, the natural ranges of lions and tigers in India overlapped. However, these two species have different habitats: lions live and breed in the open grasslands while tigers generally stay in the forest. Thus, even though lions and tigers technically can mate and produce viable offspring, this rarely, if ever occurs in natural settings.

Ecological isolation is a premating and prezygotic isolating barrier.

Temporal isolation occurs when members of two species occupy similar habitats, but breed at different times. Thus, gene flow between populations is impeded, even when the species populations occupy the same habitat within the same geographic area.

Different species of frogs, for example, may share the same pond for reproduction, but will not hybridize because they use the pond at different times of the year. Similarly, differences in the flowering times of two closely related species of plants can keep them from being cross-pollinated by one another.

Temporal isolation prevents mating between different species, and therefore also prevents the formation of a zygote, so it is a premating isolating barrier as well as a prezygotic isolating barrier.

Ethological (behavioral) isolation results from differences in courtship or mating behavior that keep members of different species from mating, even when they inhabit the same geographic area. These barriers often consist of special signals or elaborate behaviors that are used by members of a species to attract or recognize and accept mates. Examples include courtship displays (such as with the blue-footed boobies in this photo), pheromones (chemical signals), and the songs of birds.

Ethological isolation specifically prevents mating between different species and therefore also prevents the formation of a zygote. It is a premating isolating barrier as well as a prezygotic isolating barrier.

Even if there are no temporal or behavioral cues to keep individuals of two species from hybridizing, it may simply be physically impossible for mating to take place. Mechanical isolation occurs when two species have significant anatomical differences that prevent them from mating.

For example, many species of the fly genus Drosophila are virtually indistinguishable except for differences in the male and female genitalia. Similar to the workings of a lock and key, males cannot copulate successfully with females from the wrong species. .

Mechanical isolation is a premating isolating barrier as well as a prezygotic isolating barrier.

Floral isolation (pollinator isolation) occurs when gene flow between flowering plant species is reduced due to mechanical and ethological differences in pollination systems. Mechanical isolation occurs when the transfer of pollen between individuals of different species is impossible because of different flower structures. Ethological isolation occurs when pollinators preferentially visit one type of flower at the exclusion others, even if they exist in the same geographical location.

Two species of columbine provide an example of floral isolation. The two species, western columbine (Aquilegia formosa) and hairy columbine (Aquilegia pubescens) have different flower structures and different pollinators: western columbine is pollinated by hummingbirds, and the hairy columbine is primarily pollinated by hawkmoths. The two species have morphologies that have evolved to suit a particular kind of pollination. The western columbine has long, nectar-containing tubes that only can be reached by the beak of a hovering hummingbird. The hairy columbine provides a landing platform, and is fragrant in the evening to attract its moth pollinators. The two species also are ecologically isolated: the hairy columbine is generally found at higher elevations than the western columbine.

Floral isolation is a premating isolating barrier as well as a prezygotic isolating barrier.

When the gametes of one species cannot fuse with the gametes of another species to form a zygote, gametic isolation has occurred. This is a kind of "lock-and-key" isolating mechanism in which, despite successful mating or pollination, hybridization will not occur because the gametes of one species function poorly with the gametes or reproductive tract of another species.

Sea urchins provide a good example of this type of reproductive isolating barrier. Many sea urchin species live in sympatry (within the same geographic region) and shed their gametes at the same time (no temporal isolation), but remain evolutionarily distinct. In this case, the formation of hybrid zygotes is prevented because the surface proteins of the ovule (the "lock") and sperm, or male gametes (the "keys") of different species do not fit together.

Gametic isolation is a postmating isolating barrier while still a prezygotic isolating barrier.