HIF-α transcription factors, as key master regulators of oxygen homeostasis, constitute a subgroup of the large bHLH-PAS transcription factor family and have been identified in many vertebrates. Although the amino acid sequences of bHLH-PAS domain are conserved, the physiological and pathological roles of this family are variable. They also have different patterns of expression. It is possible that the HIF-α copies have been retained as a consequence of adaptive amino acid replacements or relaxed selective constraint which have conferred subtle changes in function after duplications. Phylogenetic analysis indicated that at least two major duplications had occurred early in the vertebrate lineages. Analyses of the ratios of nonsynonymous/synonymous substitution rates revealed that relaxation of selective constraints might play important roles over evolutionary time and shape variation in some members of the family. The coefficients of functional divergence (θ) estimated between pairwise comparisons of gene groups from HIF-1α, HIF-2α, and HIF-3α indicated statistically significant site-specific shift of evolutionary rates between them, suggesting that altered functional constraints may have taken place at some amino acid residues after their duplications. Moreover, we also mapped sites identified to have been relaxed from purifying selection onto the three-dimensional structure of human HIF-2α. Overall, our study demonstrated that the functional diversity of HIF-αs members may be caused by relaxed negative selection on the N-terminal transactivation domains after HIF-αs duplications, which recruited new partners leading to functional specificity.