Recently, infections with the H5N1 subtype of highly pathogenic avian influenza virus (H5N1-HPAIV) in mammals have been reported worldwide, including in cows in the United States and successive human cases in Cambodia. In Vietnam, 47 tigers and three leopards died from H5N1-HPAIV infection between August and October 2024. This study aimed to determine the origin of the H5N1 strains that infect tigers in Vietnam and to identify specific mutations associated with mammalian infection. Specimens were collected from tigers that died of suspected H5N1-HPAIV infection in southern Vietnam in September–October 2024. RNA was extracted and subjected to whole-genome sequencing. Time-stamped phylogenetic analysis was performed using H5N1 sequences recently detected in Vietnam and neighbouring countries. Phylogenetic results revealed that the strain found in tigers (Tiger H5N1 strain) belonged to clade 2.3.2.1e and was genetically close to the H5N1-HPAIV lineage responsible for ongoing human infections in Cambodia since 2023. Tiger H5N1 strains harboured several amino acid substitutions associated with mammalian host adaptation or transmissibility, such as E627?K in polymerase basic protein 2, similar to the Cambodian human H5N1 strains. This mammalian-adapted H5N1 lineage should be continuously monitored in poultry and mammals, including humans, in Vietnam to prevent further transmission.