Cephalopods are important prey in the diet of top predators, such as marine mammals and seabirds. However, detailed information on their trophic relationships in the Patagonian marine ecosystem is scarce, including those cephalopod species with commercial interest. The aims of this study were to evaluate the composition of the cephalopod component in the diet of Otaria byronia and determine the habitat use and trophic levels of their main cephalopod prey by measuring the stable isotopic signature of cephalopod beaks. Between May 2005 and February 2009, fresh faecal samples were collected from two sea lions rookeries in San Matias Gulf. Cephalopods occurred in 39.4% of the 1112 samples collected during the whole period of study. The dominant prey species was Octopus tehuelchus, which occurred in 45.8% of scats containing cephalopod remains, and represented 58.7% in terms of numerical abundance and 52.0% in mass of cephalopods consumed. The second species most consumed was the myopsid Doryteuthis gahi. The significant higher δ15N values of O. tehuelchus beaks in comparison with those of D. gahi showed that these two species have different trophic levels while occupying similar habitat (δ13C values) in neritic waters of the Patagonian shelf.