The study aimed to determine the distribution and relative abundance of freshwater fishes in and adjacent to Agulhas National Park. Fourteen fish species were collected during surveys carried out in the Heuningnes and Ratel river systems and a variety of endorheic pans from 2003 to 2005. Seven of the species are marine fishes tolerant of low salinity (Mugil cephalus, Liza richardsonii, Monodactylus falciformis, Caffrogobius gilchristi, Rhabdosargus holubi, Solea bleekeri, Gilchristella aestuaria) with the remaining seven species being primary freshwater fishes. Three are indigenous, including Pseudobarbus burchelli, Sandelia capensis, and Galaxias zebratus of which two morphological forms were recorded within Agulhas National Park. The remaining four species are invasive aliens (Cyprinus carpio, Lepomis macrochirus, Micropterus salmoides, Micropterus punctulatus). Classification using physico-chemical variables indicated four major wetland groups, with fishes occurring predominantly in the group comprising palustrine, lacustrine and riverine wetlands, and in one instance in a brackish endorheic pan. Management actions which should facilitate conservation of indigenous fishes in the Agulhas region are suggested.