Major histocompatibility complex (MHC) class I alleles of nonhuman primates have been associated with disease susceptibility, resistance, and resolution. Here, using high-resolution pyrosequencing, we characterized MHC class I transcripts expressed in Mauritian cynomolgus macaques (MCM), a nonhuman primate population with restricted MHC diversity. Using this approach, we identified 67 distinct MHC class I transcripts encoded by the seven most frequent MCM MHC class I haplotypes, 40 (60%) of which span the complete open reading frames. These results double the number of MHC class I sequences previously defined by cloning and Sanger sequencing of cDNA-PCR products and provide a rapid, high-throughput, and economical method for MHC characterization. Overall, this approach significantly expanded our knowledge of MCM haplotypes and will facilitate future studies on disease pathogenesis and protective cellular immunity.