We examined nucleotide sequence variation in centromeric histone H3 (CENH3) in fifteen rye species and subspecies, including annuals, perennials, self-pollinating and cross-pollinated plants. Levels of genetic variation within N-terminal tail (NTT) and histone fold domain (HFD) were estimated as average per site pairwise nucleotide diversity (p) and as the number of segregating polymorphic sites (S). A comparison of nucleotide diversity (ptot) for NTT and HFD showed that estimates of diversity are consistently greater in NTT. Our estimates of NTT nucleotide diversity for perennial Secale strictum C. Presl were similar to those observed in annual S. cereale L., elevating those estimates for ancient species S. silvestre. A similar pattern of nucleotide polymorthism was observed between the self-pollinating and cross-pollinated subspecies. These results suggest that lower nucleotide diversity within CENH3 for some wild and cultivated subspecies of Secale may be attributable to narrow ecological niche, either evolutionary age or breeding.