Prezygotic reproductive isolation can evolve quickly when sexual selection drives divergence in traits important for sexual interactions between populations. It has been hypothesized that standing variation for male/female traits and preferences facilitates this rapid evolution and that variation in these traits is maintained by male–female genotype interactions in which specific female genotypes prefer specific male traits. This hypothesis can also explain patterns of speciation when ecological divergence is lacking, but this remains untested because it requires information about sexual interactions in ancestral lineages. Using a set of ancestral genotypes that previously had been identified as evolving reproductive isolation, we specifically asked whether there is segregating variation in female preference and whether segregating variation in sexual interactions is a product of male–female genotype interactions. Our results provide evidence for segregating variation in female preference and further that male–female genotype interactions are important for maintaining variation that selection can act on and that can lead to reproductive isolation.