The nonstructural protein (NS1) genes of eight H9N2 subtype influenza virus strains isolated from diseased chickens on different farms during 1996 - 2001 were amplified and sequenced. The nucleotide and deduced amino acid sequences of NS1 genes of these isolates were compared. The results showed that the homologies of nucleotide and amino acid of the isolates were 96.5% - 99.5% and 94.5 - 98.6%, respectively. These indicated that NS1 genes of H9N2 influenza viruses isolated in China were well conserved. Comparison of the amino acid sequences of NS1 genes of these isolates with those of H9N2 viruses isolated in Hong Kong of China, Korea and Pakistan demonstrated that nonstructural (NS1) proteins of the eight strains had a deletion of 13 amino acid residues at the carboxy terminus. Phylogenetic analyses showed NS1 genes of these isolates belonged to the same lineage and derived from an early chicken H9 virus isolated in 1994. The NS1 genes of the eight isolates belong to different sublineages from those of the H9N2 viruses isolated in Hong Kong, Korea and Pakistan, suggesting that the geographical distribution plays a significant role in the evolution of the H9N2 subtype influenza viruses.