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Horizontal starch-gel electrophoresis was used to obtain baseline genetic information on critically endangered Pseudobarbus quathlambae populations in the Senqunyane, Jordane and Bokong Rivers in the Lesotho Highlands. Allelic frequencies were similar between the populations and generations studied. However, allele classes at a large number of polymorphic loci deviated from expected proportions, probably as a result of inbreeding, brought about by isolation in upper catchment streams. Average heterozygosity values range from 4.2–6.2% and the average genetic distance between the populations is 0.008. Gene flow between populations and between generations is good and the populations in the study area can be regarded as a single large population.