The major histocompatibility complex (MHC) class II DRB, DQB, DPB, and DOB gene clusters are shared by different eutherian orders. Such an orthologous relationship is not seen between the β genes of birds and eutherians. A high degree of uncertainty surrounds the evolutionary relationship of marsupial class II β sequences with eutherian β gene families. In particular, it has been suggested that marsupials utilize the DRB gene cluster. A cDNA encoding an MHC class II β molecule was isolated from a brushtail possum mesenteric lymph node cDNA library. This clone is most similar to Macropus rufogriseus DBB. Our analysis suggests that all known marsupial β-chain genes, excluding DMB, fall into two separate clades, which are distinct from the eutherian DRB, DQB, DPB, or DOB gene clusters. We recommend that the DAB and DBB nomenclature be reinstated. DAB and DBB orthologs are not present in eutherians. It appears that the marsupial and eutherian lineages have retained different gene clusters following gene duplication events early in mammalian evolution.