Thirty protein-coding loci of southern African black rhinoceros (Diceros bicornis) from four isolated populations were studied using starch gel electrophoresis and polyacrylamide gel electrophoresis. Gene diversity estimates varied between 0.036 and 0.058, with the Zambezi Valley population having the largest amount of protein variation. These levels are higher than those in other studies of genetic variation in black rhinoceros and are similar to the amount of genetic variation observed for outbred natural populations that are not genetically depauperate. Because the observed levels of genetic variation vastly exceed the expectations for current effective population sizes, the current levels apparently reflect large black rhinoceros populations which have existed until recently. Observed levels of genetic variation within populations are consistent with the expectations when recent demographic events are taken into account.