Trophic position(TP) of organisms is fundamental for assessing and describing food web structure. Compound-specific nitrogen isotope analysis of amino acids (AA-CSIA) may be a promising tool to estimate more precisely and accurately the TP of marine organisms than the traditional methods, specifically the highly migratory pelagic species. The jumbo squid (Dosidicus gigas) is a pelagic cephalopod endemic to the eastern Pacific Ocean. In this study, AA nitrogen isotopic values (δ¹⁵N)of squid muscle tissues were identified and compared among three sampling locations, and the TPs were also evaluated. Results showed significant differences in AA δ¹⁵N values among squid muscle tissues from different areas. The threonine, however, had lowest δ¹⁵N values, which might be due to its special nitrogen isotope fractionation mechanisms.The spatial heterogeneity of AA δ¹⁵N values was mainly influenced by proline, glutamic acid,aspartic acid, serine and lysine, indicating the variety of dietary habits and isotopic baseline values. In addition, utilizing the isotopic differences between three and more AAs to estimate TP might be more accurate than only using glutamic acid and phenylalanine. The present study provided a beneficial reference for further research on foraging ecology in pelagic cephalopod.