The continuous variation of the seasonal influenza viruses, particularly A(H1N1)pdm09, persistently threatens human life and health around the world. In local areas of southwest china, the large time-scale genomic research on A(H1N1)pdm09 is still insufficient. Here, we sequenced 45 whole-genome sequences of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018, by next-generation sequencing technology to characterize molecular mechanisms of their origin and evolution. Our phylogenetic analyses suggest that the A(H1N1)pdm09 strains circulating in Lincang belong to clade 6B and the subclade 6B.1A predominates in 2018. Further, the strains in 2018 possess elevated evolutionary rate as compared to strains in other years. Several newly emerged mutations for HA (hemagglutinin) in 2018 are revealed (i.e., S183P and R221K). Intriguingly, the substitution R221K falls into the RBS (receptor binding site) of HA protein, which could affect antigenic properties of influenza A(H1N1)pdm09 viruses, and another substitution S183P near to RBS with a high covering frequency (11/14 strains) in 2018 is exactly located at the epitope B. Notably, the NA (neuraminidase) protein harbors a new mutation I23T, potentially involved in N-glycosylation. Based on the background with a higher evolutionary rate in 2018 strains, we deeply evaluate the potential vaccine efficacy against Lincang strains and discover a substantive decline of the vaccine efficacy in 2018. Our analyses reaffirm that the real-time molecular surveillance and timely updated vaccine strains for prevention and control of influenza A(H1N1)pdm09 are crucial in the future.