In order to understand the characterization and genetic mutations of the NS1 gene of the H9N2 and H5N1 subtype avian influenza viruses(AIV),the nucleotide sequences of NS1 regions of 12 strains of AIV from 2000～2003 in southern China were tested. The NS1 genes were amplified by RT-PCR and inserted into the pMD 18-T vector, then the recombinant plasmids were transformed into competent DH5α. The sequence analysis demonstrated that the NS1 genes of H9N2 subtype contained 654 bp and encoded 217 amino acids, and the NS1 genes of H5N1 subtype contained 678 bp and encoded 225 amino acids. The homologies of amino acid sequences of NS1 protein between H9N2 were 97.7%～94.9％, and the homologies of amino acid sequences of NS1 protein between H5N1 were 99.1%～97.8％. The H9N2 and H5N1 subtype isolates were closer to some AIV strains prevalent in southern China and Hong Kong in recent years. The results require further studying the role of the NS1 protein in the evasion of the host innate defense and potential contribution to pandemic influenza.