The Largetooth Sawfish Pristis pristis is a highly threatened euryhaline elasmobranch that in recent times has undergone a significant range contraction. It now only remains in a few areas with northern Australia being the main stronghold. Previous work using a single mitochondrial gene approach suggested the existence of regional barriers to gene flow in northern Australia. In this study, whole mitochondrial sequences of 92 P. pristis from seven river drainages across northern Australia were used to refine the population structure. This approach revealed barriers to gene flow at a scale as fine as between adjacent river drainages. Except for those flowing into the Gulf of Carpentaria, all river drainages appeared to host a genetically distinct population. The apparent genetic homogeneity in the Gulf is probably due to freshwater connectivity between river drainages, either during the last glaciation event when the Gulf was a freshwater lake or through contemporary wet season flooding. These results suggest that each river drainage across the species’ range should be considered a discrete management unit unless there is evidence of freshwater connectivity. More broadly, the improved resolution of population structure obtained with whole mitogenome analysis compared to single mitochondrial gene approaches, suggests that female reproductive philopatry may have been overlooked in previous studies of some elasmobranch species.