Population genetics is a powerful tool to increase the understanding of animals that may otherwise be difficult to study, such as the platypus (Ornithorhynchus anatinus). Focusing on two adjacent river systems in New South Wales, we used 12 polymorphic microsatellite loci to investigate the population dynamics of the platypus. We found that individual river systems acted as discrete population units. Evidence of migration, presumably overland, between systems explains how these units remain connected. This establishes an isolation-by-distance pattern that maintains species continuity across most of the mainland distribution. This improved understanding of population structure will be a valuable contribution to designing accurate management plans for the long-term conservation of this unique Australian animal.