The SPF breeding program at the Caribbean Primate Research Center supplies Indian-origin rhesus macaques of known genetic and virologic background for biomedical research. In this study, population genetic analyses using 14 short tandem-repeat sequences showed that the SPF colony has remained genetically homogenous over time, with sufficient amounts of heterozygosity and minimal stratification from its founders. Intergenerational studies indicated that an average of 7 alleles have been retained, inbreeding levels have remained low, and the degree of Indian ancestry is one of the highest among several national primate research centers. The relative low genetic diversity in the free-ranging population as well as in the captive SPF and conventional colonies when compared with that of other primate centers indicates that the free-ranging population, from which the captive-colony animals were derived, has experienced significant founder effects and genetic drift during the years after its establishment. This study supports the historical origin of the free-ranging population and confirms the high value of this resource for biomedical research. Current genetic diversity levels within the SPF colony can be ensured with the practice of colony management approaches such as equalizing male:female ratios in each SPF breeding group and increasing breeding group sizes. Introducing new Indian-origin macaques from other captive colonies might help to maximize the genetic diversity of the breeding stock. Furthermore, genetic estimates must be used to rank breeders according to their genetic value or their genome uniqueness to increase founder-genome representation and curb future genetic bottlenecks and allele loss.