Classic and contemporary trophic ecology-based studies have shown that most non-native freshwater fish species (NNS) that integrate into novel environments have the potential to influence the recipient ecosystems’ structure and function. However, the interspecific trophic interactions amongst co-occurring NNS within invaded systems remain poorly studied. Here, we used carbon (δ13C) and nitrogen (δ15N) stable isotope analyses to examine general fish trophic diversity patterns (native and non-native fishes) and to explore trophic niche patterns amongst co-occurring NNS within a flow-modified river system, the Great Fish River (South Africa). The system was characterised by isotopic variation, which revealed spatial differences in trophic complexity from uninvaded headwater tributaries to invaded mainstem and downstream sections. Two of the invaded sections, the upper mainstem of the Great Fish River (UGFR) and the Koonap River, had low isotopic overlaps between NNS and the native fish assemblages. Furthermore, co-occurring NNS in these two invaded sections had variable isotopic niche sizes and low interspecific isotopic niche overlaps, suggesting the potential for trophic differentiation. By comparison, there was evidence of high resource use patterns among NNS within the lower mainstem section of the Great Fish River (LGFR), which likely reflected trophic plasticity. Overall, results of this study provided evidence of both trophic niche differentiation (UGFR and Koonap River) and niche overlap (LGFR) as probable mechanisms of co-occurrences of the non-native fishes within different invaded sections of the Great Fish River system, and underscores the difficulties associated with predicting their trophic impacts.