Sex chromosomes provide a useful context for the study of the relative importance of evolutionary forces affecting genetic diversity. The human Y chromosome shows levels of nucleotide diversity 20% that of autosomes, which is significantly less than expected when differences in effective population size and sex-specific mutation rates are taken into account. To study the generality of low levels of Y chromosome variability in mammalian genomes, we investigated nucleotide diversity in intron sequences of X (1.1–3.0 kb) and Y (0.7–3.5 kb) chromosome genes of five mammals: lynx, wolf, reindeer, cattle, and field vole. For all species, nucleotide diversity was found to be lower on Y than on X, with no segregating site observed in Y-linked sequences of lynx, reindeer, and cattle. For X chromosome sequences, nucleotide diversity was in the range of 1.6 × 10−4 (lynx) to 8.0 × 10−4 (field vole). When differences in effective population size and the extent of the male mutation bias were taken into account, all five species showed evidence of reduced levels of Y chromosome variability. Reduced levels of Y chromosome variability have also been observed in Drosophila and in plants, as well as in the female-specific W chromosome of birds. Among the different factors proposed to explain low levels of genetic variability in the sex-limited chromosome (Y/W), we note that selection is the only factor that is broadly applicable irrespective of mode of reproduction and whether there is male or female heterogamety.