Globin mRNAs of Xenopus borealis and Xenopus tropicalis have been cloned and sequenced. The nucleotide and derived amino acid sequences were compared with each other and with already available data from Xenopus laevis. This analysis rendered clear evidence that the common ancestor of X. laevis and X. borealis, but not of X. tropicalis, had lost one amino acid of the beta-globins prior to a genome duplication event that preceded the segregation of the former two species. Replacement-site substitutions were used to calculate a rough time scale of genome duplication and species segregation. The results suggest an ancient separation between the X. laevis and the X. tropicalis groups occurring approximately 110-120 million years ago. Analysis of the amino acid chains demonstrated various alterations. However, some functional domains, like heme-binding sites and alpha 1 beta 2 contact sites, were subject to a high degree of conservation, indicating the existence of functional constraints on them also in the genus Xenopus.