The Berg River redfin (Pseudobarbus burgi) is a critically endangered endemic cyprinid from South Africa. We investigated mitochondrial DNA control region variation among specimens representative of five populations drawn from two adjacent river systems. Phylogenetic analyses, a minimum spanning network, and an analysis of molecular variance underscore the pronounced genetic separation of redfins originating from the geographically closely allied Verlorevlei and Berg Rivers, two populations that may have remained isolated since the Pleistocene. Despite a lack of geographic structuring within the Berg River, historic female gene flow among the upper and middle/lower parts of the river appears to be limited and the contemporary populations are probably isolated due to deterioration of the mainstream of the river. Our results suggest that the Berg and Verlorevlei populations should be managed as distinct conservation units. We encourage the use of sanctuaries, particularly by private landowners within both river systems, as this approach may contribute effectively to preserving genetic diversity within the species.