Fish populations in upland freshwater streams tend to be highly susceptible to anthropogenic impacts such as land degradation and their isolation from other potentially suitable habitats, combined with the fragmented, dendritic structure of headwaters renders their dispersal to more suitable habitats an unlikely response to recover from such impacts. The most northern population of river blackfish (Gadopsis marmoratus) is isolated to a tiny area in the headwaters of the Condamine River in the northern Murray Darling Basin. Local knowledge reveals they were once widespread and abundant in all headwaters, and relatively common in lowland reaches. Their massive range contraction coincided with widespread land-clearing in the catchment for agriculture leading to highly degraded riparian and in-stream habitat. To gain an understanding of the remnant population of this fish we sampled them across 15 sites for an understanding of their current range then undertook an analysis of their genetic structure and variation across the sampling area. Strong genetic structure was detected among subpopulations, using microsatellites (0.173, p<0.0001) and mtDNA (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 three clusters, however these 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 their remaining habitat become unsuitable. Low effective population size and genetic diversity also suggests that local adaptation to future impacts such as altered temperature regimes under climate change is unlikely. It is likely that significant investment in restoration of the degraded lands will be required to ensure the persistence of river blackfish in the Condamine River catchment.