During the early stages of speciation, interspecific gene flow may be impeded by deleterious epistatic interactions in hybrids, which maintain parental allelic combinations at the speciation genes. The resulting semipermeable nature of the barrier to interspecific gene flow provides a valuable framework to identify the genes involved in hybrid mortality or sterility, as well as the evolutionary mechanisms that initially caused their divergence. The two Atlantic eels Anguilla anguilla and A. rostrata are partially isolated sister species that naturally hybridize, but whose genetic basis of postzygotic isolation remains unknown. We collected high-throughput sequencing data from the transcriptomes of 58 individuals and discovered 94 genes showing differentially fixed mutations between species. Evidence for positive selection at nuclear diagnostic genes was obtained using multilocus extensions of the McDonald-Kreitman test with polymorphism data from each species. In contrast, mitochondrial protein-coding genes experienced strong purifying selection and mostly diverged at synonymous sites, except for the mt-atp6 gene, which showed an atypically high nonsynonymous to synonymous rate ratio. Nuclear-encoded protein interactors of the mt-atp6 gene in the ATP synthase complex were significantly overrepresented in the list of nuclear diagnostic genes. Further analysis of resequencing data showed that positive selection has operated at both the mt-atp6 gene and its nuclear interactor atp5c1. These findings suggest that a cytonuclear incompatibility caused by a disruption of normal ATP synthase function in hybrids contributes to partial reproductive isolation between European and American eels.