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.