Southern bluefin tuna (SBT) are a highly migratory species. Each year SBT swim seasonally through the Indian Ocean, to their feeding grounds in the Great Australian Bight (GAB), South Australia where juveniles are resident during austral summer months. Large-scale marine seismic surveys over the past decade have indicated changes in SBT distribution. Since 2012, SBT have not yet returned to their historic feeding grounds and instead are found further east, towards Encounter bay, but with an inconsistent distribution. To date, we know nothing about what they feed upon during their stay in South Australian waters, particularly in areas of their new distribution. This study used a multiple evidence-based approach with biochemical tracers (e.g., stable isotopes, fatty acids) to determine the diet of SBT in South-East Australia. Results revealed wild caught SBT diet is highly variable across their distribution in South Australia and diets of captive SBT from tuna farming pens were distinct from wild caught locations. Additionally, important fatty acids for human health were detected in higher concentrations in captive SBT. These results have contributed to our understanding of SBT diet in South Australia and highlight the importance of the tuna farming industry from a human nutrition perspective.