Pigtailed macaques (Macaca nemestrina) provide an important model for biomedical research on human disease and for studying the evolution of primate behavior. The genetic structure of captive populations of pigtailed macaques is not as well described as that of captive rhesus (M. mulatta) or cynomolgus (M. fascicularis) macaques. The Washington National Primate Research Center houses the largest captive colony of pigtailed macaques located in several different housing facilities. Based on genotypes of 18 microsatellite (short tandem repeat [STR]) loci, these pigtailed macaques are more genetically diverse than captive rhesus macaques and exhibit relatively low levels of inbreeding. Colony genetic management facilitates the maintenance of genetic variability without compromising production goals of a breeding facility. The periodic introduction of new founders from specific sources to separate housing facilities at different times influenced the colony's genetic structure over time and space markedly but did not alter its genetic diversity significantly. Changes in genetic structure over time were predominantly due to the inclusion of animals from the Yerkes National Primate Research Center in the original colony and after 2005. Strategies to equalize founder representation in the colony have maximized the representation of the founders’ genomes in the extant population. Were exchange of animals among the facilities increased, further differentiation could be avoided. The use of highly differentiated animals may confound interpretations of phenotypic differences due to the inflation of the genetic contribution to phenotypic variance of heritable traits. Am. J. Primatol. 74:1017-1027, 2012. © 2012 Wiley Periodicals, Inc.