Ruffed lemurs (Varecia variegata and Varecia rubra) are categorized as Critically Endangered on the IUCN Red List, and genetic studies are needed for assessing the conservation value of captive populations. Using 280 mitochondrial DNA (mtDNA) D-loop sequences, we studied the genetic diversity and structure of captive ruffed lemurs in Madagascar, Europe and North America. We found 10 new haplotypes: one from the European captive V. rubra population, three from captive V. variegata subcincta (one from Europe and two from Madagascar) and six from other captive V. variegata in Madagascar. We found low mtDNA genetic diversity in the European and North American captive populations of V. variegata. Several founder individuals shared the same mtDNA haplotype and therefore should not be assumed to be unrelated founders when making breeding recommendations. The captive population in Madagascar has high genetic diversity, including haplotypes not yet identified in wild populations. We determined the probable geographical provenance of founders of captive populations by comparison with previous studies; all reported haplotypes from captive ruffed lemurs were identical to or clustered with haplotypes from wild populations located north of the Mangoro River in Madagascar. Effective conservation strategies for wild populations, with potentially unidentified genetic diversity, should still be considered the priority for conserving ruffed lemurs. However, our results illustrate that the captive population in Madagascar has conservation value as a source of potential release stock for reintroduction or reinforcement projects and that cross-regional transfers within the global captive population could increase the genetic diversity and therefore the conservation value of each regional population.