In 2024, we identified and sequenced 52 avian influenza A (H9N2) virus strains in Laos. Using the established H9N2 genomic classification system, a novel HA gene clade of the A/chicken/Beijing/1/94-like (BJ/94-like) lineage, designated Clade 4.6.20, was identified. This new clade is phylogenetically distinct from the previously described clades, and the representative strains in this new Clade 4.6.20 presented a low cross reactivity to the antisera of other clades, suggesting antigenic drift of the viruses between the new Clade 4.6.20, and other clades in the dominant lineage of Clade 4.6. In addition, all the newly identified viruses in Clade 4.6.20 possessed HA-L226 and NP-N52 mutations, which are associated with human-type receptor binding and human MxA-related innate immunity escape, respectively. Our findings underscore the necessity of global surveillance network and cooperation to monitor the evolution of AIVs, update vaccine seed strains, and develop new vaccines with high effectiveness against H9N2 AIVs circulating globally, which threaten poultry and human health.