Upland freshwater habitats support populations that are especially susceptible to anthropogenic change. Furthermore, their isolation from other suitable habitats, and the fragmented, dendritic structure of headwaters make dispersal an unlikely response to change. We investigated genetic structure and variation in the northernmost population of Gadopsis marmoratus, which is isolated in a tiny area in the headwaters of the Condamine River catchment, in the Murray - Darling Basin, Australia. Strong genetic structure was detected among subpopulations based on microsatellites (FST 5 0.173, p < 0.0001) and mitochondrial (mt)DNA (FST 5 0.369, p < 0.05). Effective population size was low, ranging between 18.8 and 48.2, depending on the estimation method used. Bayesian clustering revealed 3 genetic clusters, but they were not congruent with drainage patterns, suggesting a complex history of dispersal among headwaters that are isolated by waterfalls. Overall, these results suggest that G. marmoratus is unlikely to disperse into new habitats if local conditions become unsuitable. Low effective population size and genetic diversity also suggest that local adaptation would be unlikely.