Leontopithecus chrysomelas, the Golden-headed Lion Tamarin (GHLT), is an endangered and endemic Neotropical primate from the Atlantic Forest of Brazil that has suffered a reduction of its habitat and population size in the wild. Ex situ populations have been established as a relevant alternative to safeguard the species and retain its genetic diversity and evolutionary potential. This study evaluated the genetic diversity and structure of the two main Brazilian captive populations of GHLT, which have been under human care at the Primatology Center of Rio de Janeiro (CPRJ) and the Zoological Park Foundation of São Paulo (FPZSP). Our results revealed levels of genetic diversity overall comparable to those observed for other Leontopithecus species and for ex situ and in situ populations of GHLT previously studied. Bayesian and principal coordinate analyses showed a moderate differentiation between CPRJ and FPZSP populations. Both populations presented observed heterozygosity values higher than expected heterozygosity values for most of the microsatellites used in this study, suggesting that the management has been efficient in avoiding an increase in homozygosity. However, simulations point to a significant loss of genetic diversity in the next 100 years, mainly in the FPZSP population. Such data are relevant for further decision-making on the metapopulation management of L. chrysomelas in captive conditions and for integrating in situ and ex situ conservation plans.