Abstract: Objective To understand the evolutionary characteristics of neuraminidase gene of influenza A (H1N1) pdm09 virus isolated in Zhejiang province from 2009 to early 2013. Methods Viral RNA was extracted from 29 strains of influenza A (H1N1) pdm09 virus. Neuraminidase gene was amplified by RT-PCR and the ORF sequence was assembled together by using sequencing data. Neuraminidase genes from 22 representative domestic and overseas strains during 2009-2012 were downloaded from GenBank database for analysis. All the sequences were used for the construction of phylogenetic tree by using MEGA 5.1 software. Results The 1410 bp ORF sequences of neuraminidase gene were obtained by analyzing sequencing Results of the 29 strains. After alignment, high homology was observed among all the NA genes of the strains isolated all over the world. The homology between strains isolated in Zhejiang during 2009-2012 and the representative strain of A/California/04/2009(H1N1) from North America was as high as 98.20%-99.50%. The homology between the 4 strains isolated in Zhejiang in late 2012 and early 2013 and A/California/04/2009(H1N1) strain was 98.63%-99.14%. A H275Y mutation in neuraminidase protein which could confer resistance to oseltamivir was observed in an A (H1N1) pdm09 strain isolated in January 2010 through nucleic acid sequence analysis. Conclusion Although more mutations were accumulated in NA genes in the strains isolated in Zhejiang in late epidemic stage, high homology of98.20% was observed in all the NA genes. A H275Y mutation in neuraminidase protein which could confer resistance to oseltamivir was observed.