Recognition and rejection of heterospecific male gametes occurs in a broad range of taxa, although the complexity and redundancy of mechanisms underlying this postmating cryptic female choice is poorly understood. In plants, the arena for these interactions is the female reproductive tract (pistil), within which heterospecific pollen tube growth can be arrested via active molecular recognition. Unilateral incompatibility (UI) is one such pistil-mediated barrier in which pollen rejection occurs in only one direction of an interspecific cross. We investigated the genetic basis of pistil-side UI between Solanum species, with the specific goal of understanding the role and magnitude of epistasis between UI QTL. Using heterospecific introgression lines (ILs) between Solanum pennellii and S. lycopersicum, we assessed the individual and pairwise effects of three chromosomal regions (ui1.1, ui3.1, and ui12.1) previously associated with interspecific UI among Solanum species. Specifically, we pyramided ui12.1 with each of ui1.1 and ui3.1, and assessed the strength of UI pollen rejection in pyramided (double introgression) lines, compared to single introgression genotypes. We found that none of the three QTL individually showed UI rejection phenotypes, but lines combining ui3.1 and ui12.1 showed significant pistil-side pollen rejection. Furthermore, double introgression lines that combined different chromosomal regions overlapping ui3.1 differed significantly in their rate of UI, consistent with at least two genetic factors on chromosome three contributing quantitatively to interspecific pollen rejection. Together, our data indicate that loci on both chromosomes 3 and 12 are jointly required for the expression of UI between S. pennellii and S. lycopersicum suggesting that coordinated molecular interactions among a relatively few loci underlying the expression of this postmating prezygotic barrier. In addition, in conjunction with previous data, at least one of these loci appears to also contribute to conspecific self-incompatibility, consistent with a partially shared genetic basis between inter- and intraspecific mechanisms of postmating prezygotic female choice.