Strong contrasts in ambient isotope ratios and in diet suggest stable isotopes in the otoliths of oceanic fish can resolve water masses and geographic areas, promising a powerful multivariate approach for examining population structure and provenance. To test this, whole otoliths were taken from Patagonian toothfish (Dissostichus eleginoides) sampled off the Patagonian Shelf and South Georgia, on either side of a population boundary, and otolith δ18O and δ13C values were measured to see if they could distinguish South American-caught fish from those taken in the Antarctic. Values of δ18O and δ13C predicted capture area with 100% success, validating their use for distinguishing provenance and corroborating the prior evidence of population isolation. Values of δ18O in the otoliths reflected ambient values as well as seawater temperature: low values in Patagonian Shelf fish were consistent with exposure to Antarctic Intermediate Water (AAIW), and high values in South Georgia fish were consistent with exposure to Circumpolar Deep Water (CDW). In contrast, differences in otolith δ13C appeared to reflect diet: relative depletion of δ13C at South Georgia compared to the Patagonian Shelf were most likely linked to differences in sources of metabolic carbon, as well as δ13C in dissolved inorganic carbon (DIC) of seawater. These contrasting properties strongly suggest that stable isotopes can resolve the provenance of toothfish from Antarctic sampling areas that hitherto have been difficult to separate. These results show that, by using the chemistry recorded in otoliths, researchers can exploit biogeochemical variation in fully marine environments to examine the spatial ecology of oceanic fish.