High pathogenicity avian influenza virus (HPAIV) has significantly impacted upon avian and mammalian populations across the Antarctic region. All viruses detected have been genotype B3.2 with phylogenetic analyses indicating multiple independent incursions from continental South America to, and transmission between, sub-Antarctic islands. From a zoonotic perspective, several isolates contained markers of mammalian adaptation in PB2 with functional characterisation of mutants demonstrating efficient replication in primary human airway epithelial cell cultures, demonstrating that these PB2-mutations alone contributed to enhanced polymerase activity in human cell lines. No mammalian-adaptive mutations were detected in the haemagglutinin or neuraminidase genes, with viruses retaining avian receptor binding preferences. Antigenic characterisation demonstrated cross-reactivity with existing pre-pandemic candidate vaccine strains and all viruses remained susceptible to licensed frontline antiviral therapeutics. We demonstrate a complex evolving viral ecology in the sub-Antarctic region involving both avian and marine mammal hosts, with significant implications for regional wildlife populations and zoonotic risk.