Carcharodon carcharias is a protected species occupying the apex of most marine foodwebs where they are present. Aggregation areas, such as Guadalupe Island, México,  that have been related to feeding events, are of special interest for this species conservation. The aim of this study was to describe the fatty acid profile of C. carcharias' muscle for the first time on Guadalupe Island, using non-lethal biopsy methods to determine ontogenetic and sex differences in relation to diet and habitat use. Fatty acid profiles and biomarkers from different individuals are explored as a source of integrated information of their diet. Analysis of the fatty acid composition of individuals with varying total lengths (2.3-5.0 m) suggested a dietary shift between juveniles and adults occurring at aproximately 3 m. Fatty acid biomarkers indicated a higher degree of carnivorism in adults than in juveniles. Additionally, these ecological tracers suggested that juveniles feed in shallow waters close to the coast, while adults feed in deep waters along inshore and offshore areas. This study represents a first step towards using fatty acid composition as a relevant tool for further understanding dietary shifts and habitat use throughout the ontogeny of C. carcharias. However, to corroborate this, further studies with larger sample sizes are required.