We previously reported that peripheral lymphocytes from about 12% of cynomolgus monkeys lacked reactivity with anti-rhesus monkey CD3 monoclonal antibody (FN18). The nucleotide sequence analysis of the genes encoding CD3 component proteins revealed that a single amino acid substitutions found in the CD3epsilon chain determined the phenotype. In this study, we attempted to develop a method based on the restriction fragment length polymorphism (RFLP) and apply it for determination of the genotypes of individual monkeys. Comparison of the phenotype determined by fluorescence-activated cell sorter analysis with the genotype determined by RFLP analysis revealed that the FN18 -positive trait was dominant over the FN18-negative trait. It was also revealed that allele frequency was significantly different among macaques depending on the geographical region where their ancestors were derived from.