NYD-SP12 is a recently identified spermatogenesis-related gene with a pivotal role in human testis development. In this study, we analyzed between-species divergence and within-species variation of NYD-SP12 in seven representative primate species, four worldwide human populations, and 124 human clinical subjects. Our results indicate that NYD-SP12 evolves rapidly in both the human and the chimpanzee lineages, which is likely caused by Darwinian positive selection and/or sexual selection. We observed significant interpopulation divergence among human populations, which might be due to the varied demographic histories. In the association analysis, we demonstrated significant frequency discrepancy of a synonymous sequence polymorphism among the clinical groups with different sperm traits.