Specimens of the onychoteuthid squid Moroteuthis ingens were collected from four sites in the Southern Ocean: Macquarie Island, the Falkland Islands, the Chatham Rise (New Zealand) and the Campbell Plateau (New Zealand). Spatial variations in diet among these areas were investigated using stomach contents and lipid and fatty acid profiles. Myctophid fish were prominent prey items at all sites, and the diet at New Zealand sites contained temperate myctophid species that were not identified at other sites. The diet at the Falkland Islands differed considerably from other sites due to the large proportion of cephalopod prey that had been consumed by M. ingens. This is likely to be due to the absence of key myctophids, such as Electrona carlsbergi, and the abundance of smaller squid such as Loligo gahi and juvenile M. ingens over the Patagonian Shelf. Stomach contents data could not be used effectively to determine dietary differences between the Chatham Rise and Campbell Plateau, largely due to differences in sample sizes between these sites. Lipid class and fatty acid profiles of the digestive gland indicated that the diet of M. ingens differed significantly between the Chatham Rise and Campbell Plateau, despite the relative proximity of these sites. We conclude from total lipid content that this was due to a reduction in food availability to M. ingens at the Campbell Plateau. The highly productive waters of the Subtropical Front pass over the Chatham Rise, whereas the Campbell Plateau is situated in less productive sub-Antarctic water. Differences in oceanographic conditions are likely to have driven dietary variations between these two sites.