This study investigates the genetic variation of H9N2 subtype avian influenza virus (AIV) in Yunnan Province to provide a basis for further research and efforts in prevention and control. Reverse transcription polymerase chain reaction (RT-PCR) was employed to detect AIV nucleic acids in environmental samples collected in parts of Yunnan from 2020 to 2023. And the positive samples were subjected to chicken embryo virus isolation experiment. Positive samples underwent chicken embryo virus isolation, and hemagglutinin (HA) gene fragment of 28 isolated H9N2 AIV strains were amplified and sequenced. Bioinformatics software was used for nucleotide and amino acid homology analysis, receptor binding site analysis, antigenic site identification, and mapping of potential glycosylation sites, as well as constructing a phylogenetic tree. Phylogenetic analysis revealed that the HA gene of the 28 isolates clustered within the Eurasian lineage, Group I, represented by the Y280 lineage, and were closely related to the vaccine strain A/chicken/Jiangsu/WJ57/ 2012, with nucleotide homology ranging from 90.63% to 100% and amino acid homology from 92.45% to 100%. Compared with the earliest vaccine strain A/chicken/Beijing/1/1994 (H9N2) isolated in China, the isolates exhibited lower nucleotide homology (84.18%~85.68%) and amino acid homology (85.73%~88.72%). All 28 isolates had the HA protein cleavage site PSRSSR↓GLF, and mutations were detected at key receptor binding sites, including A198T/V, T163N, N232H, Q234L, and Q235M. Notably, the Q234L mutation was associated with the binding characteristics of human influenza receptors. Mutations were found at antigenic sites 234, 285, and 334, while other sites remained relatively conserved. The isolates contained 7~8 potential glycosylation sites, with deletions at positions 218 and 492, and additions at positions 313 and 491. These findings suggest that the 28 H9N2 AIV isolates were low pathogenic avian influenza viruses, with human susceptible loci. A certain degree of genetic variation was observed compared to reference strains, indicating the need for enhanced surveillance and monitoring of H9N2 AIV mutations.