Stable isotopes are a useful tool for understanding diet and migration in squid, which often play an important role in marine food webs. The Patagonian long-finned squid fishery around Falkland Islands waters is managed separately for each of its biannual fishing seasons and 2 sub-areas of the fishing zone. However, factors driving differences in the timing of recruitment and squid size between sub-areas have never been resolved. To investigate whether seasonal and spatial variation in life history parameters of the Patagonian long-finned squid Doryteuthis gahi is driven by differences in diet, stable isotope profiles (δ13C and δ15N) were measured along the gladius— the internal chitinous shell—to reconstruct a chronological record of habitat and feeding variations and to compare fishing seasons and sub-areas using a mixed modelling approach. Here we show that isotopic niche was distinct between the fishing seasons, but negligible differences in isotopic niche were found between sub-areas of the fishing zone. This suggests that spatial differences in life history parameters are not a result of dietary variation. Substantial variation in δ13C and δ15N values along the gladius indicated versatility in feeding behaviours and dietary habits over time. Squid in the second fishing season occupied a lower trophic level (mean = 3.2) than the first season (mean = 3.5). Patterns in δ13C values indicated more complex and substantial migration patterns in the second fishing season. This coincides with the current knowledge of this species’ spawning migrations. We anticipate that these data may be used for future studies investigating the role of squid within the Patagonian Shelf marine ecosystem. Analysis of the squid gladius has applications for other loliginid squid species in future studies.