Marine trophodynamic studies have been developed mainly through the evaluation of stomach contents. However, these studies only reflect the feeding of a few days, showing a "snapshot" of the food consumed for a short time. Currently, new complementary techniques have been developed to evaluate the diet of predators using biochemical markers (fatty acids, FAs) and can, in turn, consider the rate of renewal of these biochemical constituents in body tissues. In the jumbo squid Dosidicus gigas, an important fishery resource in the Humboldt Current System, we identified and analyzed the stomach contents (prey) of D. gigas collected along the coasts of Chile in order to compare the FA profiles of its prey with those of three of its principal tissues and/or organs (digestive gland, gonad and mantle muscle). The analyses of the stomach contents indicate that D. gigas feeds on fish, crustaceans and squids, as previously have been reported. Regarding the FAs, the digestive gland presented the highest concentration and diversity of FAs, followed by the gonad and then the mantle. In turn, when comparing the FAs of the digestive gland of D. gigas with the FA profiles of its preys, the crustacean Pleuroncodes monodon and the fish Lampanyctus sp. presented the closest similarity, especially with a high contribution of essential FAs and PUFAs. Our findings indicate that during the cold season (austral winter), individuals of D. gigas may present an energy optimization strategy, characterized by the intake of prey with a high energy content rich in PUFAs, such as crustaceans and small fishes. This study not only contributes to the understanding of the squid biology and their trophodynamic, but also has important implications to improve fishery management within an ecosystem approach. This is relevant because D. gigas and its prey have suffered heavy exploitation, with a significant reduction in their biomass.