The onset of reproductive barriers, or hybrid sterility, is a means of reinforcing genetic incompatibilities between isolated populations, leading to speciation. A handful of genes that underlie hybrid sterility have been discovered, but their molecular mechanism is generally unclear. Bayes and Malik reveal that the hybrid sterility gene Odysseus-site Homeobox (OdsH), which encodes a satellite-DNA binding protein, shows differential DNA binding patterns between the species Drosophila mauritiana and D. simulans; OdsH derived from D. mauritiana localizes to the D. simulans Y chromosome, whereas OdsH derived from D. simulans does not. The incorrect expression, localization, and endurance of the OdsH protein appear to affect spermatogenesis in hybrid males, explaining the mechanism underlying hybrid sterility between these species. Thus, genetic incompatibilities between satellite-DNA and their binding proteins may represent a general basis for hybrid sterility in animals.