During 2022–2024, a highly pathogenic avian influenza virus (HPAIV) H5N1 strain, designated A/Seagull/Hebei/qhd6/2024 (H5N1), was isolated from migratory birds in Beidaihe National Wetland Park, North China. Phylogenetic analyses revealed that its hemagglutinin (HA) gene belongs to the 2.3.4.4b clade, while the neuraminidase (NA) gene and internal genes clustered with strains originating from multiple continents, consistent with a transcontinental reassortment event. The virus also exhibited 90.1–98.1% nucleotide homology with human-derived H5N1 isolates. Molecular characterization identified key virulence-associated mutations, including the classic HPAIV HA cleavage site, HA-T160A (associated with enhanced human receptor-binding capacity), and NA-I117T (potentially linked to drug resistance). BALB/c mouse infection experiments confirmed systemic replication and high pathogenicity of strain qhd6, with a 50% lethal dose (LD50) of 0.95 log10EID50/mL. Antigenic analysis revealed good cross-reactivity with the widely used H5-Re14 vaccine strain. This study reports the identification, in Beidaihe National Wetland Park, of an HPAIV H5N1 strain whose genetic characteristics suggest intercontinental reassortment and indicate cross-species transmission risk. It clarifies the genetic characteristics and pathogenicity of this strain, providing an important theoretical and practical basis for precise surveillance, risk early warning, and comprehensive prevention and control of AIV at migratory bird stopover sites in North China.