Dispersal patterns can have a major impact on the dynamics and viability of populations, and understanding these patterns is crucial to the conservation and management of a species. In this study, patterns of sex-biased dispersal and waterway/overland dispersal are investigated in the endemic Australian platypus, Ornithorhynchus anatinus, a semi-aquatic monotreme. Analyses of over 750 individuals from south-eastern Australia at 13 microsatellite loci and two mitochondrial genes, cytochrome b and cytochrome oxidase subunit II, provide genetic insight into dispersal patterns. For the first time, platypuses of western Victoria are shown to be genetically distinct from other populations of the mainland. Despite distinct morphological differentiation either side of the Great Dividing Range, populations remain genetically similar between coastal and inland areas suggesting gene flow is likely to occur across these ranges. Landscape genetic analyses indicate variability in dispersal patterns between Victorian and Tasmanian platypuses with a greater avoidance of overland travel indicated in Victoria compared to Tasmania. Females appear to remain within their natal area or return to breed, maintaining greater genetic structure in maternally inherited mitochondrial DNA in comparison to nuclear DNA and sharing genetic similarity within a short river distance (i.e. ≤1.4 km). The results of this study provide a valuable spatial framework for the management of wild platypus populations within south-eastern Australia and a baseline for future monitoring of populations that are likely to be impacted by environmental and anthropogenic change.