Freshwater ecosystems show substantial loss of biodiversity as a result of anthropogenic induced stressors. In this study, we evaluated the distribution of freshwater fishes in the Kowie River catchment using historic fish distribution records supplemented by field survey data. Fishes were collected using a multi-method approach: seine nets, fyke nets and gill nets. Historic fish distribution data showed that 22 freshwater fishes from 11 families historically occurred, while in this study, we recorded a total of 16 freshwater fish species from 9 families. Overall, a decrease in the number of native species was recorded with a total of five species absent and two new non-native species recorded during the current survey. Coptodon rendalli constituted a new record in the Kowie River catchment while Clarias gariepinus was recorded for the first time in the mainstem of the Kowie River. The presence of these two non-native species in the Kowie River catchment may have implications for the conservation and management of the freshwater diversity in the catchment.