The African Penguin (Spheniscus demersus) has suffered population declines and is listed in the IUCN Red List as Endangered. The species is endemic to the coast of southern Africa, and breeding colonies are distributed on the south-western coast of Africa. Currently, African Penguins are being kept in zoo and aquarium facilities throughout South Africa. In this study, molecular genetic data based on 12 microsatellite markers from 1 119 African Penguin samples from four facilities were generated in order to determine the level of genetic variation, population structure and differentiation, and effective population size to assist in the development of an effective captive management plan. Expected heterozygosity ranged from 0.57 to 0.62, and allelic richness from 4.2 to 5.1. However, based on differences between first- and second-generation captive birds, we conclude that the ex situ population is at risk of losing genetic variability in the future and management programmes should include exchange of birds between captive facilities in order to induce gene flow and increase effective population size. Adding individuals from in situ populations should also be considered in the future in cases where these birds cannot be rehabilitated. Molecular genetic analyses of wild penguin populations should be carried out for comparison, and to ascertain to what degree ‘in situ genetic diversity’ is represented among ex situ populations. With regular resampling and analyses, the extent of the effect of processes such as genetic drift on diversity in the ex situ penguin populations will become evident.