The H9N2 subtype of the avian influenza virus (AIV) is widely prevalent in birds, threatening the poultry industry and providing genetic material for emerging human pathogens. The prevalence and genetic characteristics of H9N2 in Yunnan Province, China, are largely unknown. Samples were collected from live poultry markets (LPMs) and breeding farms in Yunnan Province. H9N2-positive samples were identified by polymerase chain reaction (PCR), with a high positivity rate of 42.86% in tissue samples. The positivity rate of swab samples in the LPMs in Kunming was 3.97% (17/564), but no AIV was detected in samples from poultry farms in Lijiang, Wenshan, and Yuxi. Evolutionary analysis and genotyping were performed for the 17 strains of isolated H9N2 virus. Phylogenetic analysis revealed that all H9N2 viral genes had 91.6%-100% nucleotide homology, belonged to the G57 genotype, and had high homology with H9N2 viruses isolated from Guangdong and Guangxi, suggesting that the H9N2 viruses in Yunnan Province may have been imported by chicks. Using a nucleotide divergence cutoff of 95%, we identified ten distinct H9N2 genotypes that continued to evolve. The surface genes of the H9N2 isolates displayed substantial genetic diversity, highlighting the genetic diversity and complexity of the H9N2-subtype AIVs in Yunnan. Molecular analysis demonstrated that all 17 strains of H9N2 isolates had mutations at H183N, Q226L, L31P, and I268V in hemagglutinin; S31N in matrix protein 2; and no replacements at positions 274 and 292 of the neuraminidase protein. Sixteen strains had the A558V mutation and one strain had the E627V mutation in polymerase basic protein 2. Analysis of these amino acid sites suggests that H9N2 influenza viruses in Yunnan continue to mutate and adapt to mammals and are sensitive to neuraminidase inhibitors but resistant to adamantanes. It is necessary to strengthen surveillance of AIV H9N2 subtypes in poultry and LPMs in Yunnan to further understand their genetic diversity.