Sympatric closely related species have developed different behavioral adaptation strategies owing to the pressure of evolutionary selection. Investigating the trophic niche patterns of these species is vital for understanding their competition and coexistence mechanisms, and is a basic problem of population dynamic. In this study, the jumbo flying squid (Dosidicus gigas) and purple back flying squid (Sthenoteuthis oualaniensis), which have sympatric distribution in tropical eastern Pacific, were selected as research objects. The trophic niches and interspecific relationships of these two species were analyzed using biogeochemical tracers (stable isotope and fatty acid). The results showed that the compositions of several essential fatty acids (C18: 2n6, C20: 2n6, C20: 3n3, C20: 4n6 and C20: 5n3) were different in muscle tissues between D. gigas and S. oualaniensis, indicating the potential differences in food sources. However, the carbon and nitrogen isotopic values did not differ between these two species, probably because they had similar trophic levels and were collected from the same area. These results are in good consistent with the observed trophic niches. The stable isotopic trophic niches exhibited a higher degree of overlap than that of fatty acid trophic niches, indicating that the compositions of fatty acids may reflect the dietary differences of sympatric closely related species more robust. The study fosters our understanding of the adaptative feeding behavior during the evolution of cephalopods, and provide an alternative approach for improving authenticity evaluation of the trophic niches and relationships among closely related species.