Objective:To analyze the genetic characterization of hemagglutinin and neuraminidase genes of influenza A/H1N1 (09pdm) viruses in Jiangsu province, 2013. Methods: Specimens from influenza-like cases in Sentinel hospitals from Jiangsu province were isolated by cell cultivation and performed by subtype identification. Fourteen representative positive strains of A/H1N1 (09pdm) viruses were selected within different regions in 2013. HA and NA genes of 14 isolates were sequenced and the genetic characterization was analyzed. Results: Compared with vaccine strain A/California/07/2009(H1N1), the HA and NA genes of 14 isolates shared the highest nucleic acid sequence similarity (97.6%~98.4% and 98.4%~98.9%, respectively), and amino acid sequence similarity (96.5%~98.0%, 97.0%~98.5%, respectively). The phylogenetic analysis showed that the HA and NA genes of all 14 strains were divided into two clusters. The amino acid substitutions of the HA proteins (D114N,K300E and E516K) and NA proteins (N44S) of 14 isolates were observed. The evolution dynamics revealed that the genetic diversity of influenza A/H1N1(09pdm) viruses increased. The positive pressure sites were observed in HA proteins site 179, 180, 239, 301, 303, 310, 311, 312, 313 (including site 310) and NA proteins 4, 23, 52, 287, 374 (including site 4) by FEL and REL model. Of 14 isolates, the HA protein contained 9 potential glycosylation sites(7 in HA1 and 2 in HA2), whereas NA protein contained 9 potential glycosylation sites. Conclusion: In 2013, the influenza A/H1N1 (09pdm) viruses had undergone molecular evolution to generate genetic diversity and this emphasize the importance of reinforcing virus surveillance.