Background: Hox genes code for homeodomain-containing transcription factors that function in cell fate determination and embryonic development. Hox genes are arranged in clusters with up to 14 genes. This archetypical chordate cluster has duplicated several times in vertebrates, once at the origin of vertebrates and once at the origin of gnathostoms, an additional duplication event is associated with the origin of teleosts and the agnanths, suggesting that duplicated Hox cluster genes are involved in the genetic mechanisms behind the diversification of vertebrate body plans, and the origin of morphological novelties. Preservation of duplicate genes is promoted by functional divergence of paralogs, either by subfunction partitioning among paralogs or the acquisition of a novel function by one paralog. But for Hox genes the mechanisms of paralog divergence is unknown, leaving open the role of Hox gene duplication in morphological evolution. Results: Here, we use several complementary methods, including branch-specific dN/dS ratio tests, branch-site dN/dS ratio tests, clade level amino acid conservation/variation patterns, and relative rate ratio tests, to show that the homeodomain of Hox genes was under positive Darwinian selection after cluster duplications. Conclusion: Our results suggest that positive selection acted on the homeodomain immediately after Hox clusters duplications. The location of sites under positive selection in the homeodomain suggests that they are involved in protein-protein interactions. These results further suggest that adaptive evolution actively contributed to Hox-gene homeodomain functions.