Speciation is responsible for the vast diversity of life, and hybrid inviability, by reducing gene flow between populations, is a major contributor to this process. In the parasitoid wasp genus Nasonia, F2 hybrid males of Nasonia vitripennis and Nasonia giraulti experience an increased larval mortality rate relative to the parental species. Previous studies indicated that this increase of mortality is a consequence of incompatibilities between multiple nuclear loci and cytoplasmic factors of the parental species, but could only explain ∼40% of the mortality rate in hybrids with N. giraulti cytoplasm. Here we report a locus on chromosome 5 that can explain the remaining mortality in this cross. We show that hybrid larvae that carry the incompatible allele on chromosome 5 halt growth early in their development and that ∼98% die before they reach adulthood. On the basis of these new findings, we identified a nuclear-encoded OXPHOS gene as a strong candidate for being causally involved in the observed hybrid breakdown, suggesting that the incompatible mitochondrial locus is one of the six mitochondrial-encoded NADH genes. By identifying both genetic and physiological mechanisms that reduce gene flow between species, our results provide valuable and novel insights into the evolutionary dynamics of speciation.