Abstract: Objectives To understand the genetic characteristics and mutation of 1 avian influenza A (H7N9) virus strain isolated in Lishui. Methods The sample was collected from a confirmed H7N9 virus infection case and the virus was cultured on MDCK cells in P3 laboratory. By using RT-PCR, the 8 RNA fragments of the cultured virus were sequenced and analyzed with bio-software. The sequences of 8 RNA fragments were multi-aligned with sequences downloaded from GISAID and GenBank. The gene characteristics and molecular mutation of key evolution-determined nucleotide sites were analyzed. Phylogenetic analysis was performed based on the phylogenetic trees. Results One human H7N9 virus strain, A/Zhejiang/LS01/2014(H7N9), was successfully isolated and whole genome was sequenced. The results showed that there was only 2 basic amino acids in cleavage site of HA, 3 mutations (A134T, G186V and Q226L) occurred in the crucial sites related with the receptor of HA protein, 1 mutation (E627K) occurred in PB2 protein, and also 1 drug resistance site (S31N) was found in M2 protein. The LS01 strain's HA protein and NA protein shared 98.90% homology with that of A/chicken/Zhejiang/DTIDZJU01/2013(H7N9) strain and 98.60% homology with that of A/chicken/Changzhou/C08/2013(H9N9) strain, respectively. Phylogenetic analyses showed that other proteins were highly homologous (99.10-99.89%) with avian H9N2 viruses. The results showed that the isolated LS01 strain was directly evolved from avian H7N9 virus, which had circulated in Shanghai, Zhejiang and Jiangsu. The avian flu viruses with high similarity were also found in adjacent regions. Conclusion The characteristics of 8 RNA fragments of LS01 strain was consistent with avian-originated H7N9 subtype virus. The HA cleavage sites had the properties of low pathogenic avian influenza virus, but mutations of Q226L and E627K might be related with high pathogenicity to human beings.