When resources are limited or patchy, a species may develop some degree of resource partitioning to reduce intra-specific competition. Development of intra-specific resource partitioning is more pronounced in species with clear phenotypic variation among individuals (e.g. age or sex). Southern elephant seals Mirounga leonina have pronounced sexual dimorphism and range widely in size and foraging range between juvenile and adult stages. However, hypothesized diet-based resource partitioning has been less clear due to difficulties in sampling diet while seals are away from breeding islands. We analysed fatty acids (FAs) from blubber of 122 juvenile seals and compared them to FA profiles from blubber of 52 adult females, and to FA profiles from 51 prey species (grouped as fish and squid) to examine evidence for diet-based resource partitioning in the seals. FA signature analysis revealed physiological and dietary differences between ages. Principle components of the 21 FAs from seal blubber and prey parts distinguished prey from seals, and clearly separated prey species into fish and squid classes. FA profiles from adult females differed to those from juveniles, with the former more 'squid-like' and the latter more 'fish-like'. Variation in FA profiles of seals was also apparent between sexes and during different seasons. Differences in diet between juveniles and adult females suggest resource partitioning occurs in response to large metabolic and physiological differences with age that limit juvenile dispersal and diving abilities. By consuming a different suite of prey species relative to adult females, juvenile southern elephant seals may reduce intra-specific competition.