This technical report is aimed at providing policy makers, fisheries managers, and conservation agencies with information on the population structure of a threatened shark of northern Australia, the Speartooth Shark. The identification of population boundaries is key to determining the appropriate spatial scale for the conservation and management of wildlife. The major river drainages (Wenlock River, Alligator Rivers, Adelaide River) comprising the species’ known range have been shown previously to be distinct genetic populations. Recent surveys have revealed a wider range than previously documented with newly-identified populations in the Daly River of the Northern Territory and the Ord River of Western Australia as well as the species’ rediscovery in Papua New Guinea (PNG). Here we aimed to test the hypothesis that the newly identified rivers (Daly and Ord Rivers), along with the Kikori River in southern PNG, also represent distinct populations given their isolation from known populations. The analysis revealed that the Ord and Kikori Rivers represent distinct populations. Results from the Daly River also suggest that this is a distinct population, although sample size was small and power limited to infer statistical significance. Each river system within the range of the Speartooth Shark should be treated as a separate management unit.