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At present there are only two populations of the vulnerable Treur River barb, Barbus treurensis, in existence; a founder population in the upper Blyde River and a translocated population in the Treur River where the species became extinct. The translocated population was derived from individuals from the upper Blyde River. We used starch gel-electrophoresis to compare the genetic structure of these two populations. Polymorphism was detected at 34% of the loci and the average heterozygosities were 0.048 for the translocated population and 0.062 for the founder population. Deviations of observed genotype frequencies from expected proportions occurred at seven loci and were the result of a deficit of heterozygotes in all instances. Estimates of population differentiation and gene flow between populations suggest that the two populations are experiencing genetic drift. This is reflected by a number of possibly unique alleles in each population, as well as alteration of common alleles at the EST-1 locus. The results indicate that the translocation affected the genetic structure of the translocated population and we recommend continued genetic monitoring of the populations as part of an effective conservation management plan.