Giant tortoises once thrived throughout the Galápagos archipelago, but today three island populations are extinct, only one individual survives from the island of Pinta, and several populations are critically endangered. We established the geographic origin of 59 captive tortoises housed at the Charles Darwin Research Station in the Galápagos Islands in an effort to find a mate for the sole survivor from Pinta (‘Lonesome George’) and to augment the number of breeders in other imperilled populations. By comparison with an extensive database of mtDNA control region (CR) haplotypes and nine microsatellites, we determined the geographic and evolutionary origin of the captive individuals. All individuals had CR haplotypes and multilocus microsatellite genotypes identical to or closely related to known haplotypes from natural populations. No obvious mate was found for Lonesome George, although we found several captive individuals carrying an evolutionarily close but geographically distinct mtDNA haplotype. Tortoises with mtDNA haplotypes closely related to another at-risk population (San Cristóbal) were also identified. These individuals could be considered as candidates for augmentation of natural populations or captive-breeding programmes and exemplify how molecular techniques can provide insights for the development of endangered species management plans.