An improved understanding of the biological and numerical properties of measures of population differentiation across loci is becoming increasingly more important because of their growing use in analyzing genome-wide polymorphism data for detecting population structures, inferring the rates of migration, and identifying local adaptations. In a genome-wide analysis, we discovered that the estimates of population differentiation (e.g., F(ST), θ, and Jost's D) calculated for human single-nucleotide polymorphisms (SNPs) are strongly and positively correlated to the position-specific evolutionary rates measured from multispecies alignments. That is, genomic positions (loci) experiencing higher purifying selection (lower evolutionary rates) produce lower values for the degree of population differentiation than those evolving with faster rates. We show that this pattern is completely mediated by the negative effects of purifying selection on the minor allele frequency (MAF) at individual loci. Our results suggest that inferences and methods relying on the comparison of population differentiation estimates (F(ST), θ, and Jost's D) based on SNPs across genomic positions should be restricted to loci with similar MAFs and/or the rates of evolution in genome scale surveys.