The Cape Fold Ecoregion (CFE) is one of southern Africa’s unique aquatic ecoregions and its freshwater fish fauna is characterized by high levels of endemism. As with many other Mediterranean-type ecosystems, the region is also a hotspot for threatened and range-restricted freshwater fish. Many of the CFE’s endemic species are at risk for extinction, with declines in population sizes and distribution ranges. The Clanwilliam sandfish Labeo seeberi is an example of such a species and is considered one of South Africa’s most endangered large migratory cyprinids. This species is endemic to the Olifants/Doring river system in the CFE and has been subject to a major population decline, mainly as a result of invasive alien fish and adverse climate events. Little is known of the genetics of the Clanwilliam sandfish, thus this study aimed to provide basic population genetic parameters to inform future conservation interventions. Both microsatellite and mitochondrial DNA (mtDNA) markers were used to assess populations from three sites within the Olifants/Doring river system. Genetic diversity was moderate to low and did not reflect the drastic decline expected on the basis of previous relative abundance data. This is likely due to a lag effect between ecological/life history demographics (due to juvenile recruitment failures) and population genetic composition. Furthermore, there was limited genetic differentiation between the sampling locations, suggesting a single breeding population, but mtDNA haplotype distribution and slight divergence of the smaller populations does suggest that the population might have become recently fragmented. The results show that the effective population size of the current breeding population might still be sufficient to maintain evolutionary potential in the short term, which could act as a buffer until conservation strategies focusing on protecting breeding animals and maximizing juvenile survival can restore population numbers.