Hypoxanthine phosphoribosyltransferase (HPRT1) is a key enzyme in the purine salvage pathway, and mutations in HPRT1 cause Lesch-Nyhan disease. The studies described here utilized targeted comparative mapping and sequencing, in conjunction with database searches, to assemble a collection of 53 HPRT1 homologs from 28 vertebrates. Phylogenetic analysis of these homologs revealed that the HPRT gene family expanded as the result of ancient vertebrate-specific duplications and is composed of three groups consisting of HPRT1, phosphoribosyl transferase domain containing protein 1 (PRTFDC1), and HPRT1L genes. All members of the vertebrate HPRT gene family share a common intron-exon structure; however, we have found that the three gene groups have distinct rates of evolution and potentially divergent functions. Finally, we report our finding that PRTFDC1 was recently inactivated in the mouse lineage and propose the loss of function of this gene as a candidate genetic basis for the phenotypic disparity between HPRT-deficient humans and mice.