Two mitochondrial DNA regions and seven microsatellite loci were examined in Patagonian toothfish from three locations in the Southern Ocean (Macquarie Island, five collections; Heard and McDonald Islands, four collections; Shag Rocks/South Georgia area, one collection). Striking mtDNA heterogeneity was detected between the three fishing locations (FST=0.445, P<0.001), but spatial and temporal collections within the same location were not significantly different. No significant overall microsatellite differentiation between the three locations was apparent (FST=−0.009, P=0.785). However, some individual loci showed small but significant differentiation, which in each case was attributable to between rather than within-location differentiation. Greater differentiation of mtDNA can, in principle, be explained either by female philopatry and male dispersal, or by its greater sensitivity to changes in effective population size. The latter seems more likely as tagging indicates that toothfish is generally a sedentary species. The genetic heterogeneity between the three locations indicates restricted gene flow, with the fish at each location comprising independent units. Depletion in one location is therefore unlikely to be quickly replaced by immigration from another.