The significant antigenic changes of clade 2.3.2.1 H5 subtype of Avian influenza virus（AIV） have occurred. To control the potential pandemic threat of variations to poultry，in this study，two reassortant viruses，named rZJ and rYT，were generated using reverse genetics. The viruses contained modified HA and NA genes of epidemic strains A/duck/JS/ZJ/2016（H5N1）and A/Chicken/SD/YT/2015（H5N1）respectively，with the internal genes derived from A/Puerto Rico/8/34（PR8）. The reassortant candidate vaccine viruses was attenuated by removal of the multi-basic amino acid motif in the HA gene by mutation and deletion. Antigenicity analysis showed that there was significant difference between rZJ and rYT，compared with Re-6 vaccine strain. Immune efficacy tests showed that the HI antibody titers could reach 8.8 log2 and 8.9 log2，respectively，after 21 days from immunization of rZJ and rYT inactivated vaccines，showing a good immunogenicity. Immune challenge tests showed that rZJ and rYT recombinant inactivated vaccines could provide SPF chickens with 100% of the protection against homologous H5N1 viruses，and Re-6 vaccine against clade 2.3.2.1 viruses could only provide about 40% and 30% of the protection，respectively. And rZJ and rYT recombinant vaccine candidate strains using reverse genetics technology provided technical support for prevention and control of the viruses of clade 2.3.2.1.