Highly Pathogenic Avian Influenza (HPAI), particularly the H5N1 clade 2.3.4.4b strain, has continued to threaten both wild and domestic birds and mammals since its emergence in North America in 2022. As part of a Barred Owl (Strix varia) management and space-use study conducted in Northern California from 2023 to 2025, we screened 125 individuals for avian influenza virus (AIV) using serological (ELISA) and molecular (RRT-PCR) methods. One adult male, GPS-tagged in May 2024 and removed in July 2024, tested seropositive for AIV antibodies but negative for active infection by PCR. A previous blood sample from May 2024 was seronegative, suggesting exposure occurred between May and July—coinciding with the Pacific Flyway’s late spring migration period. This case represents the first reported AIV seropositive Barred Owl in California and potentially the only known HPAI-positive raptor reported in Northwestern California since the introduction of this virus in 2022. The transmission route is likely attributed to the species’ generalist diet, which includes known AIV reservoirs, such as waterfowl. These findings support multi-year surveillance efforts and offer valuable insight into AIV exposure patterns among wild raptors in remote, understudied regions.