To investigate the genetic evolution, molecular variation, and potential interspecies transmission of the H9N2 subtype avian influenza virus (AIV), this study isolated and identified viruses from lung tissue samples of diseased chickens collected from farms in Shandong Province from 2023 to 2024. Full-genome sequencing, homology analysis, phylogenetic analysis, and molecular characterization were performed. Using reverse genetics， parental strains (SD11, SD341) and a recombinant virus (re_SD341) were constructed. Two host-adaptive sites in the PA gene of the recombinant strain were mutated to the avian-like pattern (PA-R356K/N409S). The replication capacities of the three strains were evaluated in different host cell lines, and the pathogenicity of SD341 and re_SD341 was assessed in mice. The results showed that two H9N2 AIV strains were isolated from 54 samples. The isolates shared high nucleotide similarity (>90%) with domestic vaccine strains used after 2000. Phylogenetic analysis of the HA gene revealed that both isolates belonged to clade B4.7.2 within lineage B. All eight gene segments were genetically close to those of several human-derived strains, with over 90% nucleotide identity in HA, PB1, PB2 and PA genes. Molecular analysis showed that the HA cleavage site of both isolates was PSRSSR↓GL, consistent with B-lineage epidemic strains around 2020. Compared to early reference strains, both isolates had deletions in the NA stalk region and acquired a novel potential glycosylation site at position 368. The HA, PB1, PB2 and PA proteins of both isolates harbored mammalian-adaptive mutations, including K356R and S409N in PA. Biological characterization demonstrated that the parental strain SD341 exhibited significantly stronger replication in the human A549 cell line than the recombinant re_SD341 strain (P < 0.000 1), and induced notable weight loss in mice within a short time frame. These results indicate that the two H9N2 AIV isolates in this study are low pathogenic strains that have undergone varying degrees of evolutionary variation. The presence of mammalian-adaptive mutations in viral proteins increases their risk of cross-species transmission. This study provides insight into the evolution of H9N2 AIV in China and offers data support for the integrated control of avian influenza and public health preparedness.