Fatty acid biomarkers were used to investigate the feeding ecology of 17 mesopelagic fish species occurring in the Southern Ocean. Fatty acid signatures of species where little or no dietary information exists were compared to fatty acid signatures of species of known diets in order to elucidate their trophic position. Principal component analysis grouped species of known diets into two clusters with amphipods and copepods comprising the main prey species, respectively. Although the majority of species of unknown diet were grouped with either of these feeding guilds, a third cluster comprising only Gymnoscopelus bolini was identified suggesting a significantly different diet for this species. Electrona antarctica also exhibited significant changes in fatty acid signatures with size. Furthermore, discriminant analysis of the four most abundant species classified species with a 90% success rate thus validating the usefulness of fatty acid signatures when trying to resolve the trophic position of species where no or little dietary information exists.