The platypus, Ornithorhynchus anatinus is a unique, iconic mammal endemic to Australia. Despite being listed as 'common' throughout its range, platypus abundance is poorly understood. Dependence on aquatic habitats in Australia renders this species potentially vulnerable to a variety of processes including drought, climate change and habitat loss. To assist with understanding population processes, 180 individuals from Tasmania and Victoria were characterised across thirteen microsatellite loci. Large genetic differences were evident between Tasmanian and Victorian O. anatinus. Within Tasmania, high levels of allelic diversity were detected with genetic differentiation identified among some populations. Similarly, allelic diversity was high within Victorian platypuses, along with significant genetic differentiation among populations. The large genetic differences found between Tasmanian and mainland platypuses indicate long-term isolation and it is likely that the lack of past/present catchment connectedness contributes to differentiation found between populations within these regions. Understanding patterns of genetic differentiation within and between catchments will help guide future conservation management decisions for platypus.