The Patagonian toothfish, Dissostichus eleginoides, is a valuable fishery species and has a discontinuous distribution across the Southern Ocean. Identification of the genetic stock structure of toothfish would allow evaluation of the suitability of the spatial scale at which fisheries management operates. Genetic subdivision seems likely given the species distribution. Population genetics studies of this species have been performed; however, they have been limited by sample size, spatial coverage and/or the type of markers investigated. As a potential solution, we developed methods for extracting toothfish DNA from otoliths that are available in large numbers from collections held at several research institutes. Genetic differentiation between the three oceanic sectors was investigated. Four mitochondrial and four nuclear markers with multiple single nucleotide polymorphisms were sequenced by high throughput sequencing for samples from six locations. Genetic differentiation was found between three sectors with nuclear markers. However, only the Pacific sector was differentiated from other sectors with mitochondrial markers. This study demonstrates the usefulness of otolith DNA as a means of increasing sample sizes for population genetics research of fish. Additionally, the combination of nuclear and mitochondrial markers may allow insight into how the observed differences in movements between male and female toothfish impact population structure.