Cynomolgus macaques (Macaca fascicularis, Mafa), alias the crab-eating monkeys or long-tailed macaques, live across a vast range of South-East Asia. These non-human primates have emerged as important animal models in infectious and chronic diseases and transplantation studies, necessitating a more extensive characterization of their major histocompatibility complex polymorphic regions. The current information on the polymorphic variation or diversity of the Mafa-DPB1 locus is largely limited in comparison with the more commonly studied rhesus macaque DPB1 locus. In this article, to better elucidate the degree and types of polymorphisms and genetic differences of Mafa-DPB1 locus among three South-East Asian populations and to investigate how the allele differences between macaques and humans might affect their respective immune responses, we identified 40 alleles within exon 2 of the Mafa-DPB1 locus by DNA sequencing using 217 individuals. We also performed evolutionary and population analyses using these sequences to reveal some population-specific alleles and trans-species allelic conservation between the cynomolgus macaques and the rhesus macaques. Of the 40 new alleles, eight belong to a newly identified lineage group not previously found in the rhesus macaque species. This allele information will be useful for medical researchers using the cynomolgus macaques in disease and immunological studies.