The primary freshwater fish Pseudobarbus burchelli (Smith 1841) occurs across four presently isolated river systems in the south-western cape floristic region of South Africa. Mitochondrial DNA cytochrome b (701 base pairs) and control region (601 base pairs) genes were sequenced to assess the evolutionary history of P. burchelli and evaluate the role of climatic and landscape changes in shaping patterns of genetic variation in this species. We identified three historically isolated lineages in P. burchelli: a widespread lineage that occurs across three isolated river systems and two geographically restricted lineages. The results were evaluated against predictions of the confluence of river systems during low sea levels of the last glacial maximum. Occurrence of the widespread Breede lineage in the Duiwenhoks River system is consistent with reconstructed palaeoriver systems. However, the occurrence of this lineage in the Goukou river system that formed part of the eastern Gourits–Goukou palaeoriver system can only be explained by translocation or a recent river capture or episodic inundation of low drainage divides. Extreme ecological gradients or the potential presence of instream physical barriers could have prevented an exchange of lineages between the Breede and Heuningnes river systems.