Comparative evolutionary analyses of gene families among divergent lineages can provide information on the order and timing of major gene duplication events and evolution of gene function. Here we investigate the evolutionary history of the alpha-globin gene family in mammals by isolating and characterizing alpha-like globin genes from an Australian marsupial, the tammar wallaby, Macropus eugenii. Sequence and phylogenetic analyses indicate that the tammar alpha-globin family consists of at least four genes including a single adult-expressed gene (alpha), two embryonic/neonatally expressed genes (zeta and zeta'), and theta-globin, each orthologous to the respective alpha-, zeta-, and theta-globin genes of eutherian mammals. The results suggest that the theta-globin lineage arose by duplication of an ancestral adult alpha-globin gene and had already evolved an unusual promoter region, atypical of all known alpha-globin gene promoters, prior to the divergence of the marsupial and eutherian lineages. Evolutionary analyses, using a maximum likelihood approach, indicate that theta-globin, has evolved under strong selective constraints in both marsupials and the lineage leading to human theta-globin, suggesting a long-term functional status. Overall, our results indicate that at least a four-gene cluster consisting of three alpha-like and one beta-like globin genes linked in the order 5'-zeta-alpha-theta-omega-3' existed in the common ancestor of marsupials and eutherians. However, results are inconclusive as to whether the two tammar zeta-globin genes arose by duplication prior to the radiation of the marsupial and eutherian lineages, with maintenance of exon sequences by gene conversion, or more recently within marsupials.