The crosses between closely relatedDrosophilaspecies usually produce sterile hybrid males with spermatogenesis disrupted at post-meiotic phase, especially in sperm individualization stage than the pre-meiotic stage. This is possibly due to the rapid interspecies divergence of male sex and reproduction-related genes. Here we annotated 11 key spermatogenesis genes in 35 strains of species belonging tonasuta-subgroup ofDrosophila, where many interspecies crosses produce sterile males. We characterized the divergence and polymorphism in the protein coding regions by employing gene-wide, codon-wide, and lineage-specific selection analysis to test the mode and strength of selection acting on these genes.Our analysis showed signature of positive selection at bag of marbles (bam)andbenign gonial cell neoplasma (bgcn)despite the selection constrains and the absence of endosymbiont infection which could potentially drive rapid divergence due to an arms race whileroughex (rux)showed lineage-specific rapid divergence in frontal sheen complex ofnasuta-subgroup.cookie monster (comr)showed rapid divergence consistent with the possibility of meiotic arrest observed in sterile hybrids ofDrosophilaspecies. Rapid divergence observed atdon juan (dj) and Mst98Ca-likewas consistent with fused sperm-tail abnormality observed in the hybrids ofDrosophila nasutaandDrosophila albomicans. These findings highlight the potential role of rapid nucleotide divergence in bringing about hybrid incompatibility in the form of male sterility; however, additional genetic manipulation studies can widen our understanding of hybrid incompatibilities. Furthermore, our study emphasizes the importance of young species belonging tonasuta-subgroup ofDrosophilain studying post-zygotic reproductive isolation mechanisms.