The Antarctic toothfish (Dissostichus mawsoni) exhibits a circumpolar distribution in coastal waters south of the Antarctic Polar Front. For a preliminary evaluation of global population structure in this species, we examined four mitochondrial regions and 13 nuclear gene fragments in samples from four CCAMLR Subareas in the Southern Ocean (Australian Antarctic Territory (Subarea 58.4.2), Ross Dependency (Subareas 88.1 and 88.2) and the South Shetland Islands (Subarea 48.1). Significant genetic differentiation within and among locations was observed for both mitochondrial and nuclear loci. The single nucleotide polymorphism (SNP) markers developed here will be useful for more extensive analyses of population structure in this species.