Southern Ocean humpback whales Megaptera novaeangliae are capital breeders, breeding in the warm tropics/subtropics in the winter and migrating to nutrient-rich Antarctic feeding grounds in the summer. The classic feeding model is for the species to fast while migrating and breeding, surviving on blubber energy stores. Whilst northern hemisphere humpback whales are generalists, southern hemisphere counterparts are perceived as krill specialists, but for many populations, uncertainties remain regarding their diet and preferred feeding locations. This study used bulk and compound-specific stable isotope analyses and isoscape-based feeding location assignments to assess the diet, trophic ecology and likely feeding areas of humpback whales sampled in the Ross Sea region and around the Balleny Islands. Sampled whales had a mixed diet of plankton, krill and fish, similar to the diet of northern hemisphere humpback whales. Proportions of fish consumed varied but were often high (2-60%), thus challenging the widely held paradigm of Southern Ocean humpback whales being exclusive krill feeders. These whales had lower 15N values and trophic position estimates than their northern hemisphere counterparts, likely due to lower Southern Ocean baseline 15N surface water values and a lower percentage consumption of fish, respectively. Most whales fed in the Ross Sea shelf/slope and Balleny Islands high-productivity regions, but some isotopically distinct whales (mostly males) fed at higher trophic levels either around the Balleny Islands and frontal upwelling areas to the north, or en route to Antarctica in temperate waters off southern Australia and New Zealand. These results support other observations of humpback whales feeding during migration, highlighting the species’ dietary plasticity, which may increase their foraging and breeding success and provide them with greater resilience to anthropogenically mediated ecological change. This study highlights the importance of combining in situ field data with regional-scale isoscapes to reliably assess trophic structure and animal feeding locations, and to better inform ecosystem conservation and management of marine protected areas.