We analyzed the antigenic and genetic properties of 20 H9N2 viruses that have been isolated from domestic poultry in China during the last 10 years. Hemagglutination inhibition (141) assays with the antisera of five selected viruses showed that 18 of the viruses could cross-react well with the antisera that were induced by A/chicken/Shijiazhuang/2/98, A/chicken/Guangxi/10/99 and A/chicken/Shanghai/10/01, though antigenic variation among these strains were observed. Two viruses, A/chicken/Shijiazhuang/2/98 and A/chicken/Heilongjiang/35/00, could react well with the homologous antisera but poorly with others. The antisera, of A/chicken/Shandong/6/96, the current vaccine strain, and A/Chicken/Heilongjiang/35/00 could not cross-react efficiently with other viruses. Phylogenic analysis showed that 19 of 20 viruses genetically were related to the Y-280 sub-lineage of Eurasia lineage, however, A/Chicken/Heilongjiang/35/00 was closely related to the early H9N2 isolate, A/Turkey/Wisconsin/66, and went into the North American lineage. These results are the first report about the antigenic and genetic diversity that existed among the H9N2 avian influenza viruses (AIV) isolated in Mainland China. (C) 2004 Published by Elsevier B.V.