The combined fish collection databases of the Albany Museum and the J.l.B. Smith Institute of Ichthyology are used to identify hotspots of endemism and threatened fish distributions in South Africa. Hotspots of fish species richness occur in the north-eastern lowveld sectors of South Africa and along the ecotone between the tropical/ subtropical and temperate faunal zones. Hotspots of endemic fish richness occur within both the tropical and temperate faunal regions, notably in the Olifants River system, Western Cape and in areas of high relief such as the Cape Fold Mountains, the Amatola-Winterberg (Eastern Cape), and the Drakensberg Escarpment (Kwazulu/ Natal-Eastern Transvaal). Threatened taxa are concentrated in the hotspots of endemic species richness which coincide largely with areas of major river conservation concern. There is limited scope for fish conservation within the ambit of formal (or informal) declared reserves, and the survival of the fauna depends on the success of river catchment conservation management. The value of museum collections in identifying areas of conservation concern for freshwater fishes is emphasized, and highlights the importance of well-preserved voucher specimens for biodiversity conservation