To evaluate how avian influenza virus (AIV) circulates among the avifauna of the Antarctic and sub-Antarctic islands, we surveyed 14 species of birds from Marion, Livingston and Gough islands. A competitive enzyme-linked immunosorbent assay was carried out on the sera of 147 birds. Quantitative reverse transcription polymerase chain reaction was used to detect the AIV genome from 113 oropharyngeal and 122 cloacal swabs from these birds. The overall seroprevalence to AIV infection was 4.8%, with the only positive results coming from brown skuas (Catharacta antarctica) (4 out of 18, 22%) and southern giant petrels (Macronectes giganteus) (3 out of 24, 13%). Avian influenza virus antibodies were detected in birds sampled from Marion and Gough islands, with a higher seroprevalence on Marion Island (P = 0.014) and a risk ratio of 11.29 (95% confidence interval: 1.40–91.28) compared to Gough Island. The AIV genome was not detected in any of the birds sampled. These results confirm that AIV strains are uncommon among Antarctic and sub-Antarctic predatory seabirds, but they may suggest that scavenging seabirds are the main avian reservoirs and spreaders of this virus in the Southern Ocean. Further studies are necessary to determine the precise role of these species in the epidemiology of AIV.