The H9N2 avian influenza virus (AIV) is currently widespread worldwide, posing a severe threat to the poultry industry and public health. To investigate the epidemiological characteristics, genomic evolution, and mammalian pathogenicity of poultry-derived H9N2 viruses in Shandong Province, a total of 192 samples from diseased chickens were collected across 10 cities in Shandong Province between February 2021 and December 2023. Real-time quantitative PCR (RT-qPCR) detection identified 10 positive samples, from which 8 virus strains were isolated. Five representative isolates were selected for whole-genome sequencing, phylogenetic analysis, molecular characterization, and mouse pathogenicity evaluation. Infected chickens mainly showed respiratory symptoms and pathological lesions in multiple organs. The five isolates shared 91.3%–99.8% nucleotide identity and were highly homologous (98.26%–99.95%) with recent Chinese H9N2 strains. Phylogenetic results indicated multiple-lineage reassortment: PB2 and M genes belonged to the G1-like lineage, PB1, PA, NP, and NS genes to the SH/F98-like lineage, and HA and NA genes to the Y280-like lineage. All isolates carried the low?pathogenic HA cleavage site (PSRSSR/GL) and human adapted receptor binding mutations (HA Q226L, I163T, H183N), with HA glycosylation loss at position 218 but retention at 313. In addition, substitutions were observed at antigenic sites, including G90E, S145D, N167G, and T200R. All isolates carried mammalian adaptation related mutations, such as PB2 A588V, PA K356R, PA-X R195K and NS1 F103L/M106I; only the CK/SD/TA1016/22 strain contained the PB2 K526R mutation. All strains carried M2 S31N conferring amantadine resistance, and a new NA glycosylation site at position 306 was found in CK/SD/TA803/22. The pathogenicity to mice showed that the three tested isolates could replicate efficiently in the lungs without prior adaptation, but could not cause mice death. Among them, the CK/SD/TA1016/22 strain showed higher replication levels and more severe lung lesions. In conclusion, H9N2 AIV in Shandong Province continue to evolve and undergo antigenic drift, and they possess evident potential for mammalian adaptation. Continuous epidemiological surveillance and risk assessment are therefore strongly recommended.