"Classical" MHC class I (I-a) genes are extraordinarily polymorphic, but "nonclassical" MHC class I (I-b) genes are monomorphic or oligomorphic. Although diversifying (positive) Darwinian selection is thought to explain the origin and maintenance of MHC class I-a polymorphisms, genetic mechanisms underlying MHC class I-b evolution are uncertain. In one extreme model, MHC class I-b loci are derived by gene duplication from MHC class I-a alleles but rapidly drift into functional obsolescence and are eventually deleted. In this model, extant MHC class I-b genes are relatively young, tend to be dysfunctional or pseudogenic, and orthologies are restricted to close taxa. An alternative model proposed that the mouse MHC class I-b gene thymus leukemia Ag (TL) arose ∼100 million years ago, near the time of the mammalian radiation. To determine the mode of evolution of TL, we cloned TL from genomic DNA of 11 species of subfamily Murinae. Every sample we tested contained TL, suggesting this molecule has been maintained throughout murine evolution. The sequence similarity of TL orthologs ranged from 85-99% and was inversely proportional to taxonomic distance. The sequences showed high conservation throughout the entire extracellular domains with exceptional conservation in the putative Ag recognition site. Our results strengthen the hypotheses that TL has evolved a specialized function and represents an ancient MHC class I-b gene.