The rhesus macaque exhibits individual differences in susceptibility and resistance to infectious agents such as simian immunodeficiency virus (SIV) under experimental conditions, and these may be genetically determined at least in part by major histocompatibility complex (MHC) class I polymorphism. Although the importance of defining MHC class I polymorphism is well recognized, development of a generic and comprehensive molecular typing method of MHC class I alleles of the rhesus macaque has been hampered because, during the evolution of this species, multiple copies of similar DNA sequences have been generated by duplication events including the coding sequences of Mamu-A and Mamu-B loci. We report here a newly developed reference strand-mediated conformation analysis (RSCA)-based typing method of multiple Mamu-A and Mamu-B cDNAs that allowed us to estimate the number of expressed alleles. This technique detected 1-7 Mamu-A signals and 2-12 Mamu-B signals in a single sample, indicating that the number of functional alleles may vary. By comparing the data from the parents with those from the descendants in the breeding colony, several MHC class I haplotypes consisting of variable numbers of functional Mamu-A and Mamu-B alleles could be assigned.