To identify the important prey items for pre-recruit Antarctic toothfish in the SSRUs 58.4.1.C, 58.4.1.G, 58.4.1.E, and 58.4.2.E, their stable carbon and nitrogen isotope ratios were determined. Sampling efforts were made during a longline survey of pre-recruit toothfish from commercial fishing vessel. In the previous studies, similarities in total FA compositions and the FA profiles in muscle tissue of Antarctic toothfish, bycatch specimen and icefish in the stomachs indicated a trophic connection between the toothfish and icefish. In this study, similarities in the δ13C values δ15N values of Antarctic toothfish indicate they occupy same trophic position irrespective of the area, body size, sex, and gonadal maturity. 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 such as stable isotope analysis after lipid extraction, stomach contents analysis, and fatty acid analysis are needed to better understand the carbon flow through the Antarctic ecosystems.