<p>Research on regional circulation and evolution of influenza A viruses before and after the COVID-19 pandemic is crucial for informing vaccine updates and antiviral drug development. This study generated 260 new genomic sequences of influenza A(H1N1)pdm09 viruses collected in Yunnan province, China, between 2018 and 2023. Comparative genomics analyses elucidated their evolutionary characteristics and dynamics. Epidemiological analysis identified key risk factors (sex, age, occupation) for influenza infection. Phylogenetic analyses revealed the sequence divergences between the vaccine strains and Yunnan circulating strains, especially in the 2020–2024 influenza seasons. The subclade reassortment events were extremely limited among these sequenced Yunnan strains, suggesting the reassortment may be not a major contributor for the circulation and evolution of influenza A(H1N1)pdm09 viruses in Yunnan during these influenza seasons. We detected the elevated evolutionary pressures acting on the specific gene segments, reflected in increased d_N/d_S ratios, particularly for envelope proteins. Furthermore, numerous amino acid substitutions (e.g., S185I/T) within HA antigenic epitopes and receptor binding sites were identified in most Yunnan strains, indicating potential roles of antigenic drift in modulating viral antigenicity and host adaptation. Notably, 17 amino acid substitutions in HA and NA (including HA: N156K) accumulated to higher frequencies during the 2022–2023 and 2023–2024 seasons. These changes likely represented the molecular signature of contemporary A(H1N1)pdm09 viruses in Yunnan. Collectively, this study explored the molecular evolutionary dynamics of A(H1N1)pdm09 viruses in Yunnan province during diverse influenza seasons, providing new regional data for studying molecular characterization and evolution of A(H1N1)pdm09 within the global surveillance framework.</p>