The platypus (Ornithorhynchus anatinus) has a wide distribution in Australia, encompassing the southern island of Tasmania and a broad latitudinal range of the mainland from the temperate south to the tropical north. We used 12 microsatellite markers from 235 individuals sampled from 13 river systems to examine patterns of genetic differentiation and gene flow throughout the species’ range. Using a Bayesian approach we identified three large-scale groupings that correspond closely to geographically distinct regions of the species’ distribution: the tropical northern mainland, the subtropical and temperate southern mainland, and Tasmania. Six additional clusters were found within the regional groups, three in the northern, two in the southern mainland regions, and the last in Tasmania. These clusters coincided with major river drainages. Genetic differentiation was generally high, with pairwise F_st values ranging from 0.065 to 0.368 for regions and 0.037 to 0.479 for clusters. We found no evidence of contemporary gene flow among the three clusters in the north, but some migration may occur between the larger clusters in the south. Due to the high genetic structuring and lack of gene flow between these three regional populations of the platypus we recommend their treatment as evolutionarily significant units (ESUs) within the platypus species. We have also detailed several smaller management units (MUs) existing within our study area based on subregional clusters and geographically significant features.