This study aimed to investigate the genetic variation and evolutionary patterns of H9N2 subtype avian influenza viruses (AIVs) circulating in urban and rural live poultry markets in Ezhou City, Hubei Province, China in 2024. Environmental samples were collected from live poultry market surroundings and tested for H9N2 viral nucleic acids. Positive samples were subjected to virus isolation, followed by sequencing of the hemagglutinin (HA) and neuraminidase （NA） genes of the isolated strains. Bioinformatics tools were used to analyze their genetic characteristics. Phylogenetic analysis and nucleotide similarity assessments showed that the HA and NA genes of the two isolates clustered within the h9.4.2.5c and 1.2 lineages, respectively, with nucleotide similarities to early reference strains ranging from 85.57% – 91.83% (HA) and 85.51% ~ 89.29% (NA). Key amino acid site analysis revealed that the HA cleavage site was PSRSSR ↓ GLF, indicating low pathogenicity. Several mutations associated with receptor binding (T155N， S158N, F/A160N) were observed in the HA protein. In the NA protein, a deletion of three amino acids (TEI) at positions 63-65 in the stalk region and mutations at the red blood cell binding site (K/E/S368N, D369N) were identified. No known resistance-associated mutations were found in the NA gene. Evolutionary rate estimation indicated that the HA and NA genes had average substitution rates of 2.479 × 10-3 and 1.573 × 10-3 substitutions/site/year, respectively. The estimated times to the most recent common ancestor (tMRCA) were 2003 for HA and 2009 for NA, suggesting a higher evolutionary rate in the HA gene. These findings indicate that the two H9N2 isolates obtained from the outdoor environment of live poultry markets in Ezhou City harbored mutations at key functional sites that may enhance viral adaptation to mammalian hosts. This study highlights the potential for cross-species transmission of H9N2 viruses during their evolution and underscores the need for ongoing molecular surveillance to assess their public health risk and support the development of effective prevention and control strategies.