Sexual signals are critical for normal reproductive function within species and, when divergent between lineages, can also contribute to the expression of species reproductive isolation. We are examining the evolution and genetics of sexual signaling in several systems, including chemoattraction between male and female gametes in wild tomato species. Using a semi in vivo assay to directly observe interactions between pollen tubes and ovules, we show that pollen is guided to ovules via an ovule-secreted chemoattractant, and that some species pairs show reductions in pollen tube attraction to heterospecific ovules consistent with the evolution of partial isolation. We identify two cysteine-rich peptides as candidate ovule-secreted chemoattractants, and show that one candidate has fixed non-synonymous differences between species that show reduced chemoattraction. A genetic analysis using ovules from hybrid introgression lines shows that this locus induces pollen tube behavior that recapitulates con- and heterospecific patterns of gamete chemoattraction, supporting it as a strong candidate for the ovule-secreted chemoattractant. These findings address the phenotypic expression and molecular basis divergent heterospecific gamete signaling across multiple species a clade, and demonstrate how sexual signaling that facilitates intraspecific mating events can contribute to reduced sexual compatibility between lineages.