Female Amazon mollies, Poecilia formosa, are a unisexual species that reproduce by gynogenesis. They must coexist and mate with males of other species (usually the mollies Poecilia latipinna or Poecilia mexicana) to induce embryogenesis, but inheritance is strictly maternal. We examined the mating preference of the male sailfin molly, P. latipinna, for female sailfin mollies versus Amazon mollies, P. formosa. We compared the mating preferences of sympatric and allopatric populations collected throughout the Gulf Coast of North America. Male P. latipinna from six populations sympatric with Amazon mollies showed a significantly greater strength of preference for conspecific sailfin females than males from five populations that were allopatric with Amazon mollies. These results provide strong evidence for reproductive character displacement of male mate choice in sympatry. Furthermore, the large geographical range of populations that we tested revealed variation among populations within sympatry and allopatry, indicating that it is important to evaluate a large number of populations when examining reproductive character displacement.