Abstract: Objective To study the genetic variation and evolution characteristics of HA1 gene of influenza A (H3N2) virus isolated in Guangxi from 2007 to 2009 and compare the genetic differences on HA1 regions between A3 virus circulated in Guangxi and vaccine strains recommended by WHO and the strains circulating in Shanghai, Beijing and Zhejiang. Methods Viral RNA of 22 strains isolated in Guangxi during 2007 - 2009 were extracted and their HA1 genes were amplified and sequenced, and the gene sequences were analyzed with the biometric software. Results The strains isolated in 2007 were closed to the strains isolated in 2008, the homogeneity of nucleotide and amino acids between them was 99.39% and 99.08% respectively, but it was distinct to the strains isolated in 2009, the homogeneity was 99.04% and 97.78% respectively. Compared with vaccine strain A/Brisbane/10/2007 recommended by WHO for 2008 - 2009, the homogeneity of amino acids was 99.81%, 99.25% and 97.94%. The strains isolated in 2009 had replaced 6 amino acids in the antigenic determinant A, D and E. The homogeneities of outbreak strains and sporadic strains were all above 99.00%. The homogeneity of amino acids between the strains isolated in 2007 and the strains in Shanghai and in Beijing was 99.83% and 99.12% respectively, while the homogeneity of amino acids was 98.93% and 99.25% respectively in 2008. The analysis of phylogenetic tree showed that the strains isolated in 2009 was in separate branch, which was far from the strains isolated in 2007 - 2008 and the vaccine strains recommended by WHO. Conclusion The genetic characteristics of HA1 gene of influenza A (H3N2) virus isolated in Guangxi in 2007 was similar to that isolated in 2008. The strains isolated in 2009 had genetic variation. The strains isolated in Guangxi were closed to the strains isolated in Shanghai and Zhejiang, but distinct to the strains isolated in Beijing. The genetic variation of vaccine strains recommended by WHO was behind the circulating strains in Guangxi.