The Antarctic toothfish, Dissostichus mawsoni, serves as a valuable fisheries resource around the Antarctic Continent since 1997, managed by the Commission for the Conservation of Antarctic Marine Living Resources (CCAMLR). Although defining genetic or stock structure of populations is crucial for improving fishery management of this species, its number of populations or stocks and genetic diversity levels remain unclear. In the present study, we assessed the population genetic and phylogeographic structure of the Antarctic toothfish populations across 20 geographic localities spanning from the two main Subareas 88 and 58 (58.4, 58.5), based on a combined analysis of mitochondrial DNA (mtDNA) cytochrome oxidase I (COI) and 16S rRNA (16S) sequences and seven nuclear microsatellite loci. MtDNA revealed a low level of polymorphism (h=0.571, π=0.0006) with 40 haplotypes in 392 individuals, connected only by 1-5 mutational steps. Nonetheless, microsatellites showed much higher variation with allelic richness (AR) values of 6.328 (88.3 RB3) to 7.274 (88.3 RB6) within populations. Levels of genetic diversity were generally higher for the 58 Subarea populations than for the 88 Subarea. Eight of 15 populations showed a genetic signal of inbreeding, despite no sign of population bottlenecks detected. Population structure analyses of microsatellites suggest that the sampled 88 and 58 Subareas are likely to comprise a well-admixed single gene pool (one genetic stock), probably due to high contemporary gene flow during the prolonged epipelagic larval phase of this fish. However, given weak, but significant microsatellite differentiation found between six population-pairs (58.4.2 A vs 88.3 RB4; 58.5.2 vs 88.1 RBK; 58.5.2 vs. 88.3 RB1; 88.1 RBI vs 88.2 RB1; 88.1 RBI vs 88.3 RB4; 88.1 RBH vs 88.3 RB4), the possibility of existence of multiple stock lineages could not be excluded. The mtDNA AMOVA also indicated a significant difference in the population structure between the 88 and 58 Subarea groups (FCT=0.011, P=0.004). To clarify these issues, further study with additional polymorphic markers (such as microsatellites or SNPs) using more samples from other areas, particularly the 48 Subarea will greatly help to determine the population or stock structure of an entire population of D. mawsoni more concretely. The findings of this study will inform conservation efforts on the stock (unit) management for this valuable fisheries resource. Genetic monitoring for the Antarctic toothfish populations will be essential to understand how well these valuable fisheries resource will sustain in response to ongoing climate changes.