Commercial fishing is having an increasingly negative impact on marine biodiversity with over 70% of the world’s fish stocks being fully exploited and in many cases overexploited. On top of this, the Commission for the Conservation of Antarctic Marine Living Resources (CCAMLR) has granted commercial fishing permits in the most remote marine environment on earth, the high latitude Southern Ocean. The primary target of these new commercial fishing ventures is the large pelagic piscivorous predator, the Antarctic toothfish, Dissostichus mawsoni. Unfortunately little information is available on the demography, genetics or life history of this large fish. Without such information we have little idea as to the effects of commercial fishing on the population structure and survival of this species. In this study we focus on patterns of genetic diversity within and between geographically disparate populations of the Antarctic toothfish using randomly amplified polymorphic DNA (RAPD) markers. Results of our study showed high levels of genetic similarity within and between populations. Despite high levels of genetic similarity, genetic analyses detected significant population structure including fixed differences among populations, a significant fixation index (Fst) and between population differentiation via a Mantel test. From a conservation perspective, low levels of genetic diversity may be indicative of relatively small populations that would not be able to withstand heavy commercial fishing pressures. Given that there is evidence for significant genetic structure, it will be important to manage these fisheries in a manner that will help prevent the loss of unique genetic variation from regional overfishing.