The structure of the pelagic food web is a key issue to understand the variability in C and N fluxes in the ocean. The trophic structure of the hyperiid amphipods and its spatial variation in the subtropical area of the Southeast Pacific was studied based on C and N isotope composition. Zooplankton were collected off northern and Central-southern Chile during September-November 2016 at the coastal zone and in oceanic areas. Measurements of δ15N and δ13C of individual hyperiids were used to estimate the trophic position for each species, and to assess the community isotopic niche for 3 pre-defined areas: the coastal upwelling (eutrophic) zone, the coastal transition (mesotrophic) zone and the oceanic (oligotrophic) zone, as well as three depth layers. Our results showed different assemblages of species among the 3 zones, but also a large overlap between the isotopic niche for all zones and depth layers. In the mesotrophic zone there was a noticeable increase in the trophic position of individual species. The trophic diversity of hyperiids over the study region was relatively high, including primary consumers, omnivores and carnivorous feeders, and was associated with the changes in productivity across zones. However, carnivory was more prevalent in the mesotrophic zone and in the mesopelagic (500–1000 m) layer. We suggest that such variations reflect both the biogeochemical processes controlling the productivity in each zone, and the life-traits of individual species.