Freshwater habitats are some of the most imperilled ecosystems in the world as they harbour numerous species threatened with extinction. In tropical Africa, acute deficiency of scientific data on the distribution patterns of freshwater biodiversity hampers successful conservation interventions. The number of newly described and resurrected freshwater fish species in southern Africa has increased considerably since the last bioregionalization effort, nearly three decades ago.  Here, we utilize an updated matrix of catchment-scale native freshwater fish distributions to re-evaluate earlier biogeographic zonation patterns and examine the relative contribution of beta diversity to observed spatial distribution patterns in the subregion. Cluster analysis applied to an incidence data matrix of 259 native freshwater fish species from 17 drainage basins resulted in three major biogeographic zones, which generally corresponded to patterns shown in earlier studies. However, our analysis further revealed a split of the Eastern zone into two sub-clusters -- Northeast and Southeast. We decomposed the overall beta diversity (β_SOR), of southern Africa’s native freshwater fishes into its nestedness (β_SNE) and turnover (β_SIM) components. In all three zones, the proportion of the nestedness resultant component (βratio) was less than 0.5, implying that the compositional variation in overall beta diversity was mainly driven by species turnover. The dominance of the turnover component to overall β-diversity suggests that conservation initiatives targeting multiple sites across broad spatial scales are likely to provide better outcomes for southern Africa’s native ichthyofauna than a few large, protected areas. We discuss the relative contribution of environmental heterogeneity and dispersal limitation on observed bioregionalization and β-diversity patterns of native freshwater fishes in southern Africa. Incomplete knowledge of the taxonomic diversity of southern Africa’s ichthyofauna affects the mapping of distribution patterns, stressing the need for increased sampling efforts, especially in high diversity drainages that border the Congo basin.