The sperm protein associated with nucleus in the X chromosome (SPANX) genes cluster at Xq27 in two subfamilies, SPANX-A/D and SPANX-N. SPANX-A/D is specific for hominoids and is fairly well characterized. The SPANX-N gave rise to SPANX-A/D in the hominoid lineage approximately 7 MYA. Given the proposed role of SPANX genes in spermatogenesis, we have extended studies to SPANX-N gene evolution, variation, regulation of expression, and intra-sperm localization. By immunofluorescence analysis, SPANX-N proteins are localized in post-meiotic spermatids exclusively, like SPANX-A/D. But in contrast to SPANX-A/D, SPANX-N are found in all ejaculated spermatozoa rather than only in a subpopulation, are localized in the acrosome rather than in the nuclear envelope, and are expressed at a low level in several nongametogenic adult tissues as well as many cancers. Presence of a binding site for CTCF and its testis-specific paralogue BORIS in the SPANX promoters suggests, by analogy to MAGE-A1 and NY-ESO-1, that their activation in spermatogenesis is mediated by the programmed replacement of CTCF by BORIS. Based on the relative density of CpG, the more extended expression of SPANX-N compared to SPANX-A/D in nongametogenic tissues is likely attributed to differences in promoter methylation. Our findings suggest that the recent duplication of SPANX genes in hominoids was accompanied by different localization of SPANX-N proteins in post-meiotic sperm and additional expression in several nongonadal tissues. This suggests a corresponding functional diversification of SPANX gene families in hominoids. SPANX proteins thus provide unique targets to investigate their roles in the function of spermatozoa, selected malignancies, and for SPANX-N, in other tissues as well.