The distribution of a freshwater species is often dependent on its ability to disperse within the riverine system. Species with high dispersal abilities tend to be widespread, whereas those with restricted dispersal tend to be geographically restricted and are usually given higher conservation priority. Population structure was compared between a widespread freshwater prawn species, Macrobrachium australiense, and a narrow-range endemic freshwater prawn, Macrobrachium koombooloomba. The distribution of M. australiense and M. koombooloomba did not overlap, although suggested historical river-boundary rearrangements indicate that there has been the potential for dispersal into neighbouring catchments. A fragment of the mtDNA CO1 gene was analysed and a Mantel test revealed a significant isolation by distance effect for both species. Significant overall FST values confirmed that both species exhibited low levels of dispersal, a prediction for populations inhabiting a fragmented upland environment. The level of structure in M. australiense is surprising for a widely distributed species. Not all M. australiense populations conformed to the stream-hierarchy model, with results being best explained by historical river realignment or cross-catchment dispersal. The fact that both species show limited dispersal highlights the importance of conservation in highland areas for both endemic and widely spread species.