H5N1 subtype influenza virus A/Duck/Shandong/093/2004 (A/SD/04) strain was chosen as the master strain for rescue research. 11 sets of primers for 8 plasmids construction were designed base on the sequencing of the full-length of A/SD/04. Eleven fragments of A/SD/04 were amplified by the designed primers and were ligated with PHW2000 for rescue plasmid construction. Eight transcription/expression plasmids were obtained, which encoded the eight segments of A/SD/04, and designated as 241, 242, 243, 244, 245, 246, 247 and 248, respectively. The COS-1 cell was cotransfected with eight plasmids with different combination of A/SD/04 and PR8. The eight reassortants shared the same HA (from A/SD/04) but contained different internal genes and NA. All of the eight reassorted viruses had some similar bio-characteristics, such as the viral title in fertilized eggs was range from 256 to 1024, the EID50 were between 10(-8.5) to approximately 10(-9), and MDT were between 34 to approximately 46h. But the IVPI of the eight reassortants was differently and all were lower than the wild-type A/SD/04. These results confirmed that different recombination of internal genes of H5N1 has influence on viral virulence to 6-week SPF chicken but not on viral replication ability in embryonated chicken eggs. The establishment of eight-plasmid rescue system for A/SD/04 is the base for farther research on genes function of H5N1. And A/SD/04 can be used as a backbone to replace PR8 entirely in generation of H5 AIV vaccine candidate.