Objective To prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China. Methods Recombinant viruses were rescued using reverse genetics. Neuraminidase(NA) and hemagglutinin(HA) segments of the A/Xinjiang/1/2006, A/Guangxi/1/2009, A/Hubei/1/2010, and A/Guangdong/1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpolI vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their embryonate survival and antigenicity were compared with those of the respective wild-type viruses. Results The 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics. Conclusion The 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of pre-pandemic vaccines. Objective To prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China. Methods: Recombinant viruses were rescued using reverse genetics. Neuraminidase (NA) and hemagglutinin (HA) segments of the A / Xinjiang / 1/2006, 1/2009, A / Hubei / 1/2010, and A / Guangdong / 1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpolI vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their The 4 genomes were compared with those of wild-type viruses. Results The 4 recombinant viruses showed similar antigenic compared with wild-type viruses, chicken embryo survival and antigenicity were. 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of p re-pandemic vaccines.