To infer important prey resources for Antarctic toothfish (Dissostichus mawsoni) in the south of SSRUs 881.J and 881.L in the southern Ross Sea, their lipid composition was determined and compared tolipid profiles of fish and invertebrate species taken as bycatch in the fishery or collected from stomachs of toothfish. Stable carbon and nitrogen isotope ratios were also determined to further identify feeding relationships between these species. The aim of this study was to establish the feasibility of tracking main dietary items of pre-recruit Antarctic toothfish by comparing results of biomarker analysis and conventional diet analysis. Sampling collections were made during a longline survey of pre-recruit toothfish from research vessel in February 2012. Results of fatty acid (FA) and stable isotope analyses from this study provide evidence that a combination of these two techniques can delineate the main prey items of Antarctic toothfish and trophic structure of the toothfish-related fish food web in the southern Ross Sea ecosystem. Similarities in total FA compositions and the FA profiles in muscle tissue of Antarctic toothfish and Pleuragramma antarctica, Pogonophryne barsukovi, Dacodraco hunteri, and Trematomus loennbergii indicated a trophic connection between toothfish and these fish species. Meanδ15N values of Antarctic toothfish were higher than those of P. antarcticum, P. barsukovi, and T. loennbergii, indicating a higher trophic position of thetoothfish. In contrast, similar δ15N values between Antarctic toothfish and icefish (D. hunteri) suggested that they occupy the same trophic position. Overall results of this survey are consistent with the frequency and percentage occurrence of prey in Antarctic toothfish stomachs. Further collection and subsequent biomarker analyses for more pelagic and benthic biota are needed to better understand entire food web structure in the southern Ross Sea.