The giant Australian cuttlefish (Sepia apama) forms an annual breeding aggregation at Whyalla (South Australia) that comprises more than a quarter of a million individuals, a phenomenon not known in other cephalopods. An appropriate strategy for managing this species is required including knowledge on the population structure. It has been suggested that the best approach to determine population structure is to use a combination of complementary methods that address different aspects of the biology of a species. Molecular, chemical and morphometric techniques were used to determine the population structure of S. apama in South Australia. Results from the three techniques are in agreement and indicate that the breeding aggregation in Whyalla is one of two significantly different populations of S. apama in South Australia. A potential third population may exist in the region between the breeding aggregation and Gulf St Vincent, however, levels of differentiation between this population and Whyalla are different from year to year. Findings are of importance for the management of S. apama, in addition to increasing our understanding of the species dynamics and evolution. The presence of at least two populations means that S. apama in South Australia must be managed as separate populations.