A total of 31 representative strains of influenza A virus H3N2 subtype isolated at different time and regions in Nanjing between 2013 and 2014 through influenza pathogen monitoring system were selected for this study. The genetic distance between the vaccine strain A/Texas/50/2012 and the isolated group at nucleotide and amino acid levels were 0.010 2 and 0.012 1, respectively. The similarities of nucleotide and amino acid sequence were 97.9%-99.6% and 97.2%-99.3%, respectively. The phylogenetic analysis showed that hemagglutinin (HA) gene of 31 isolates could be divided into several groups. Three 2013 strains belonged to 3B, three 2014 strains belonged to 3C.1, two 2014 strains belonged to 3C.2b and the other 23 strains belonged to 3C.3a. Compared with the vaccine strain, the accumulated mutations on epitopes reached 24. The amino acid substitutions of N128A/T and P198S/A on HA in 31 isolates were observed. A new glycosylation site 126 NWT occurred in 7 isolates, and 45NSS and 144NNS glycosylation sites disappeared in 3 isolates. The results suggest that HA gene of H3N2 has a significant variation compared to the vaccine strain, and this may be related to the vaccine prevention program in Jiangsu Province during 2013-2014.