To estimate the genetic variability of two subspecies of cynomolgus monkeys (Macaca fascicularis fascicularis and M. f. aurea) using microsatellite markers, 26 microsatellite markers were selected from previous reports. Seventeen markers showed high polymorphism in a subset of monkeys and were used for the assessment of genetic diversity in the larger sample. The effective number of alleles, the polymorphism information content (PIC) and the expected heterozygosity of M. f. aurea monkeys were all statistically significantly higher than those of M. f. fascicularis monkeys (P < 0.05), suggesting the M. f. aurea monkeys had a higher degree of genetic variation than the M. f. fascicularis monkeys. Substantial differences in allele distribution were also detected between the two subspecies of cynomolgus monkeys. Private alleles restricted to the M. f. fascicularis or the M. f. aurea monkeys were found throughout the selected 17 loci. These private alleles may allow the discrimination of the two subspecies of cynomolgus monkeys. The selected markers could also be used to estimate the genetic variation for other subspecies of cynomolgus monkeys. Further work using additional animals obtained from native or independent sources will be important for a more complete understanding of the genetic differences between these two subgroups.