Abstract: The aim of this study was to develop H5 subtype avian influenza vaccine with all genes from avian origin influenza viruses, and provide a DIVA (Differentiating Infected from Vaccinated Animals) strategy for the control of avian influenza. Sequences encode several basic amino acids of HA gene from A/mallard/Huadong/S/2005(H5N1，S strain) were deleted. Using reverse genetics system, we rescued two reassortant viruses (rH5N6/F and rH5N2/F) that contained NA genes from A/duck/England/1/1956(H11N6, E strain) or A/Chicken/Shanghai/F/98（H9N2, F strain） and internal genes from F strain. The two vaccine viruses were avirulent to SPF chickens and embryonated chicken eggs, replicating well in eggs and MDCK cells, while rH5N6/F showed better growth in eggs. The results indicate that the reassortant viruses are suitable for vaccine manufacturing and being used as reference vaccine virus against the H5N1 clade 2.3.4 viruses in China.