Epistasis, the nonadditive interaction between loci, is thought to play a role in many fundamental evolutionary processes, including adaptive differentiation and speciation. Focusing on species differences in ecophysiological traits, we examined the strength and direction of pairwise epistatic interactions between target chromosomal regions from one species, when co‐introgressed into the genetic background of a foreign species. A full diallel cross was performed using 15 near‐isogenic lines (NILs) constructed between two tomato species (Solanum habrochaites and Solanum lycopersicum) to compare the phenotypic effects of each chromosomal region singly and in combination with each other region. We detected main effect quantitative trait loci (QTLs) for two of our three focal traits. Epistatic effects accounted for c. 25% of detected effects on trait means, depending on the trait. Strikingly, all but two interactions were antagonistic, with the combined effect of chromosomal regions acting in the opposite direction from that of one or both individual chromosomal regions. Our study is one of the few to systematically examine pairwise epistatic effects in a nonmicrobial system. Our results suggest that epistatic interactions can contribute substantially to the genetic basis of traits involved in adaptive species differentiation, especially highly complex, multivariate traits.