The Patagonian longfin squid Doryteuthis gahi has an annual life cycle with two seasonal cohorts (autumn and spring spawners). Previous studies estimated the trophic level of adult squid by stable isotope analysis of their muscle or gladius tissues, but few studies compared different sizes and spawning cohorts. In this study, mixed models were used to compare carbon (δ13C) and nitrogen (δ15N) stable isotope signatures of muscle and gladius tissues of the autumn and spring spawning cohorts and squid of different sizes. A published isoscape model provided δ15N baselines to calculate the trophic level of D. gahi. Sampled herbivorous prey such as Euphausiacea, planktonic juveniles Munida gregaria and salps Ihlea supported the trophic level estimates. The autumn spawning cohort had higher δ13C values than the spring spawning cohort. δ15N values increased with increasing dorsal mantle length, suggesting an ontogenetic change towards larger prey. Muscle tissue was enriched in 15N but slightly depleted in 13C compared to gladius tissue. Adult squid had an estimated trophic level of ~3.2, with no significant differences between spawning cohorts. Stable isotope values followed a seasonal trend, with lower δ15N values and lower δ13C value between July and September. Large individuals of the spring spawning cohort were found to have the least niche overlap with other groups of D. gahi. Our study of the trophic levels and ecology of D. gahi can provide important information on its role in the food chain and can build the basis of studies on interaction with other species, and the impact of environmental changes on their populations. This information can inform the development of management strategies aimed at preserving the balance of the ecosystem and ensuring its sustainable use.