Life history characteristics of Antarctic toothfish (Dissostichus mawsoni), were evaluated using otolith microchemistry techniques. Our aims were to (i) determine whether fish from in CCAMLR SSRUs 88.1C (Ross Sea) and 88.2H (Amundsen Sea), could be differentiated by the trace elemental concentrations in the otolith edge and then to (ii) assess the potential of natal signatures to distinguish stocks among these regions. For the elements (Mg, Al and Sr) spatial variation in otolith chemistry was shown. This resulted in the correct classification of 63% of fish overall to their original capture sites. Discrimination of otolith natal signatures using Al:Ca and Zn:Ca showed greater classification success (79%), compared to the otolith edge which suggested the majority of fish from SSRU 88.1C and 88.2H show similar patterns of structuring consistent with their known capture location, indicating they may have used different spawning habitats between areas. PCA analyses of natal signatures apportioned in total 83% of fish from both SSRU 88.1C and 88.2H to 88.1C. The remaining group consisted entirely of individuals from SSRU 88.2H (17%), suggesting SSRU 88.2H fish may have used different spawning habitats. However, given these analyses only included adults, further investigations using a larger sample base of both adults and juveniles collected from shelf and slope regions within SSRU 88.1 and 88.2 would provide stronger evidence of structuring between these regions.