cDNAs for Xenopus beta2-microglobulin (beta2m), the obligatory light chain of most vertebrate Major Histocompatibility Complex (MHC) class I molecules, were isolated and ESTs were identified. Alignment of the deduced amino acid sequence to other species' beta2m showed that the overall structure is evolutionarily conserved, and phylogenetic analysis showed that the Xenopus beta2m sequence is intermediate between fish and bird/mammal beta2m. The Xenopus beta2m mRNA is expressed ubiquitously with highest expression in intestine, spleen, and thymus, correlating well with classical class Ia expression. beta2m mRNA and protein were also detected in Xenopus thymic tumor and kidney cell lines. Segregation analysis on a tetraploid Xenopus laevis family revealed two independently segregating, non-MHC-linked loci. As expected, only one locus was found in the diploid Xenopus tropicalis, strongly suggesting that the two beta2m loci in the tetraploid species were generated by genome-wide duplication, and did not undergo diploidization unlike many other MHC genes.