Ex situ conservation strategies may be substantially informed by genetic data, and yet only recently have such approaches been used to facilitate captive population management of endangered species. The Galápagos tortoise Geochelone nigra is an endangered species that has benefited greatly from the application of molecular and population genetic data, but remains vulnerable throughout its range. The geographic and evolutionary origins of 98 tortoises in private collections and zoos on three continents were identified using mitochondrial DNA (mtDNA) control region sequences and multi-locus microsatellite genotype data relative to a large database of representative samplings from all extant populations, including historical population allele frequency data for the Geochelone nigra abingdoni taxon on Pinta by way of museum specimens. All but six individuals had mtDNA haplotypes previously sampled, with the novel haplotypes identified as most closely related to robust populations on the islands of Santa Cruz and Isabela. Multi-locus genotypic assignments corroborated the results obtained from the mtDNA analyses, with 83.7% of individuals consistently assigned to the same locality by both datasets. Overall, the majority of captive unknowns sampled were assigned to the La Caseta Geochelone nigra porteri population, with no fewer than six individuals of hybrid origin detected. Although a purported Pinta individual was revealed to be of Pinzón ancestry, the two females currently housed with Lonesome George exhibited haplotypic and genotypic signatures that indicate that they are among the most appropriate matches for captive breeding. More generally, molecular approaches continue to represent important tools for assessing conservation value, minimizing hybridization and guiding management programs for preserving the distinctiveness of G. nigra taxa in captivity.