Hybrid sterility is a common form of reproductive isolation between nascent species. Although hybrid sterility is routinely documented and genetically dissected in speciation studies, its developmental basis is rarely examined, especially in generations beyond the F1. To identify phenotypic and genetic determinants of hybrid male sterility from a developmental perspective, we characterized testis histology in 312 F2 hybrids generated by intercrossing inbred strains of Mus musculus domesticus and M.m. musculus, two subspecies of house mice. Hybrids display a range of histologic abnormalities that indicate defective spermatogenesis. Among these abnormalities, we quantified decreased testis size, reductions in spermatocyte and spermatid number, increased apoptosis of meiosis I spermatocytes, and more multinucleated syncytia. Collectively, our phenotypic data point to defects in meiosis I as a primary barrier to reproduction. We identified seven quantitative trait loci controlling five histologic traits. A region of chromosome 17 that contains Prdm9, a gene known to confer F1 hybrid male sterility, affects multinucleated syncytia, apoptosis of round spermatids, and round spermatid numbers.