The platypus Ornithorhynchus anatinus is an endemic monotreme species with a wide latitudinal distribution in eastern Australia, including Tasmania. Understanding of the phylogeography within this species is very limited at present and represents a gap in the documentation of Australia's unique biodiversity. We analysed mitochondrial DNA sequences (partial control region and complete cytochrome b, including portions of flanking tRNAs) of 74 individuals from across the distribution of the species. Phylogenetic analysis of the concatenated sequences corroborated the primary split within the platypus, showing two major clades: one from mainland Australia and the other from Tasmania/King Island. Estimates of divergence times suggest that these clades last shared a common mitochondrial ancestor ∼0.7–0.94 Ma. Using an extended dataset of partial control region sequences from 284 individuals, we found evidence of genetic structure between river basins, primarily within mainland Australia, as well as an additional divergent lineage in North-eastern Australia. Overall, few haplotypes were shared between river basins. Analyses of molecular variance of the control region sequences indicated low rates of gene flow and significant divergence, particularly at the river basin and geographical area scales.