Abstract: Several H4 subtype avian A influenza viruses were isolated from waterfowls in some live bird markets when we surveying the ecology of the influenza in East China for more than four years and identified by specific RTPCR. In this paper, the hemagglutinin (HA) gene and neuraminidase (NA) gene of three representative virus named A/Duck/Yangzhou/216/2002 (Dk/YZ/216/02), A/Duck/Yangzhou/526/2003（Dk/YZ/526/03）and A/Duck/Yangzhou/36/2004（Dk/YZ/36/04） had been sequenced and their pathogenicity to SPF chickens had been determined. The results showed that the HA nucleotide sequence of Dk/YZ/216/02 have high identity with Tk/Minnesota/833/80(H4N2),that of Dk/YZ/526/03 and Dk/YZ/Z36/04 have high identity with Budgerigar/Hokkaido/1/77（H4N6）; Phylogenetic analysis of the N6 NA genes revealed that the NA gene of Dk/YZ/Z36/04 had genetically close relationships with that of H4N6 viruses isolated from pigeon in jiangxi China, the NA gene of Dk/YZ/526/03 had genetically close relationships with that of H9N2 viruses isolated from duck in Hokkaido. The sequences of cleavage site between HA1 and HA2 consist of P-E-K-A-S-R in all three AIVs, coincide with that of identical low pathogenic AIV, and none of the chickens inoculated with the three AIVs displayed any obvious signs of clinical disease.