AbstractSexual isolation, a reproductive barrier, can prevent interbreeding between diverging populations or species. Sexual isolation can have a clear genetic basis; however, it may also result from learned mate preferences that form via sexual imprinting. Here, we demonstrate that two sympatric species of mice—the white-footed mouse (Peromyscus leucopus) and its sister species, the cotton mouse (P. gossypinus)—hybridize only rarely in the wild despite co-occurrence in the same habitat and lack of any measurable intrinsic postzygotic barriers in laboratory crosses. We present evidence that strong conspecific mating preferences in each species result in significant sexual isolation. We find that these preferences are learned in at least one species:P. gossypinussexually imprints on its parents, but inP. leucopus,additional factors influence mating preferences. Our study demonstrates that sexual imprinting contributes to reproductive isolation that reduces hybridization between otherwise interfertile species, supporting the role for learning in mammalian speciation.