ABSTRACT The clustered Hox genes, which encode homeodomain transcription factors, control cell fates along the anterior-posterior axis. Differences between Hox proteins cause differences between body parts. Vertebrates have 13 Hox subgroups, called paralog groups, which can be correlated with some of the insect and Amphioxus genes, and have remained distinctive for hundreds of millions of years. We identify characteristic residues that define the different paralog groups. Some paralog groups can be recognized by the homeodomain sequence alone; others only by using characteristic residues outside the homeodomain. Mapping characteristic residues onto the known homeodomain crystal structure reveals that most of the homeodomain amino acids that distinguish paralog groups are oriented away from the DNA, in positions where they might engage in protein-protein interactions.