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The utility of restriction-site associated DNA sequencing (RADseq) to resolve fine-scale population structure was tested on an abundant and vagile fish species in a tropical river. Australia’s most widespread freshwater fish, the "extreme disperser" Leiopotherapon unicolor was sampled from 6 locations in an unregulated system, the Daly River in Australia’s Northern Territory. Despite an expectation of high connectivity based on life history knowledge of this species derived from arid zone habitats, L. unicolor was not a panmictic population in the tropical lower Daly. Using similar to 14 000 polymorphic RADseq loci, we found a pattern of upstream versus downstream population subdivision and evidence for differentiation among tributary populations. The magnitude of population structure was low with narrow confidence intervals (global F-ST = 0.014; 95% CI = 0.012-0.016). Confidence intervals around pairwise F-ST estimates were all nonzero and consistent with the results of clustering analyses. This population structure was not explained by spatially heterogeneous selection acting on a subset of loci, or by sampling groups of closely related individuals (average within-site relatedness approximate to 0). One implication of the low but significant structure observed in the tropics is the possibility that L. unicolor may exhibit contrasting patterns of migratory biology in tropical versus arid zone habitats. We conclude that the RADseq revolution holds promise for delineating subtle patterns of population subdivision in species characterized by high within-population variation and low among-population differentiation.