Speciation involves the reproductive isolation of natural populations due to the sterility or lethality of their hybrids. However, the molecular basis of hybrid lethality and the evolutionary driving forces that provoke it remain largely elusive. The hybrid male rescue (Hmr) and the lethal hybrid rescue (Lhr) genes serve as a model to study speciation in Drosophilids because their interaction causes lethality in male hybrid offspring. Here, we show that HMR and LHR form a centromeric complex necessary for proper chromosome segregation. We find that the Hmr expression level is substantially higher in Drosophila melanogaster, whereas Lhr expression levels are increased in Drosophila simulans. The resulting elevated amount of HMR/LHR complex in hybrids results in an extensive mislocalization of the complex, an interference with the regulation of transposable elements, and an impairment of cell proliferation. Our findings provide evidence for a major role of centromere divergence in the generation of biodiversity.