Euryhaline and freshwater elasmobranchs are considered amongst the most threatened groups of aquatic animals. The speartooth shark Glyphis glyphis has a distribution restricted to estuaries and rivers in northern Australia and Papua New Guinea. Due to this restricted range and threats from fisheries and habitat degradation, the species is currently listed as Endangered on the IUCN Red List. To inform and direct conservation management actions, we investigated the distribution and habitat utilisation of G. glyphis in the Wenlock River system, Queensland, Australia. Using acoustic transmitters and a network of fixed underwater acoustic hydrophones, the movements of 40 sharks (63 to 131.5 cm total length) were tracked over 22 mo, throughout 137 km of river and adjacent coastal embayment. Three broad zones were utilised by tagged G. glyphis, comprising a lower (0 to 23 km), mid (20 to 50 km) and upper (35 to 68 km upstream) estuary zone. Individual-based occupancy of these zones changed with seasonal changes in freshwater inflow. These results provide new insight into the habitat requirements of G. glyphis, the importance of natural eco-hydrological flows for the species, and the significance of the Wenlock River as a G. glyphis natal area.