To identify the important prey items for pre-recruit Antarctic toothfish in the SSRUs 88.1.K of the southern Ross Sea and 58.4.1.C of the eastern Antarctic Sea, their fatty acid (FA) composition and stable carbon and nitrogen isotope ratios were determined and compared to those of bycatch species and stomach samples. Sampling efforts were made during a longline survey of pre-recruit toothfish from commercial fishing vessel in February-March 2013. Similarities in total FA compositions and the FA profiles in muscle tissue of Antarctic toothfish, bycatch specimen (Channichthyidae), and icefish in the stomachs indicated a trophic connection between the toothfish and icefish. Difference in the δ13C values of Antarctic toothfish between two areas showed their dependence on the different organic matter resources. δ15N values of Antarctic toothfish were higher than those of most specimens collected, indicating higher trophic postion of the toothfish. Similar δ15N values between two areas 88.1.K and 58.4.1.C suggested that they occupy the same trophic position irrespective of the area. The combination of stable isotope ratios and fatty acid profiles can be effectively used to trace the trophic transfer from organic matter sources to higher trophic levels through food chains. Further studies on the trophic relationship between Antarctic toothfish and other animals by collection and subsequent biomarker analyses for more pelagic and benthic biota are needed to better understand the carbon flow through the Antarctic ecosystems.