Pseudobarbus quathlambae (Barnard, 1938) is a cyprinid minnow with a disjunct distribution divided into tributaries of the Upper Orange River system in the Lesotho Highlands and the Mzimkhulu River system in KwaZulu Natal Province (KZN), South Africa. Recent records in the Mzimkhulu River system extended the species' geographic range, and this could represent the last remnant population of this species in South Africa. However, its future existence is in doubt, because of continued threats to all the remnant populations by non-native fishes, and more recently by the building of large, interconnected dams as part of the Lesotho Highlands Water Project (LHWP). One of these dams has directly affected the Mohale population in central Lesotho, which was the largest of the seven extant populations. Analysis of mitochondrial control region sequences (534 bp) revealed considerable genetic differentiation (2.3-6.4%) and geographic structuring among the remnant populations. The phylogenetic analysis identified three lineages within P. quathlambae: (i) one in KZN, (ii) a second one in eastern Lesotho, and (iii) a third one in Mohale, central Lesotho. The deep genetic divergence and pronounced geographic structuring among the populations suggests a long period of isolation, indicating they represent unique evolutionary units.