Emerging avian viruses increasingly threaten Antarctic wildlife, raising concerns about ecosystem health and biodiversity. In this study, we conducted a comprehensive investigation of avian influenza virus (influenza A virus, IAV) in both resident and migratory birds inhabiting the South Shetland Islands, Antarctica. During the 2024–2025 austral summer, 278 samples were collected and screened using real-time RT-PCR targeting the IAV M gene. IAV RNA was detected in 30 samples, and eight of these were found to be positive for H5. Complete genome sequencing was performed on samples from a gentoo penguin (Pygoscelis papua) and a southern giant petrel (Macronectes giganteus), revealing the presence of highly pathogenic avian influenza virus H5N1, clade 2.3.4.4b. Phylogenetic analysis demonstrated that these viral genomes closely cluster with contemporary South American strains, indicating a direct connectivity between Antarctic seabirds and the broader H5N1 transmission network. Our findings highlight the heightened vulnerability of Antarctic ecosystems to emerging infectious diseases and emphasize the critical need for sustained genomic surveillance. These efforts are essential to monitor wildlife health, inform conservation strategies, and implement effective biosecurity measures to safeguard Antarctic biodiversity.