H9N2 viruses have circulated in domestic poultry in Mainland China since 1994, and an inactivated vaccine has been used in chickens to control the disease since 1998. The present study analyzed 27 H9N2 avian influenza viruses that were isolated from chickens Blouson Belstaff Robert Downey and ducks from 1996 to 2002. Infection studies indicated that most of the viruses replicate efficiently but none of them is lethal for SPF chickens. However, these viruses exhibit different phenotypes of replication in a mouse Belstaff Soldes model. Five viruses, including 4 early isolates and one 2000 isolate, are not able to replicate in mice; 14 viruses replicate to moderate titers in mouse lungs and cause less than 5% weight loss, while other 8 viruses could replicate to high titers in the lungs and 7 of them induce 10–20% weight loss of the mice on day 5 after inoculation. Most of the viruses isolated after 1996 are antigenically different from the vaccine strain that is currently used in China. Three viruses isolated in central China in 1998 are resistant to adamantanes. Phylogenetic analysis revealed that all of the viruses originated from CK/BJ/1/94-like virus and Belstaff Classic Tourist Trophy Beige formed multiple genotypes through complicated reassortment with QA/HK/G1/97-, CK/HK/G9/97-, CK/SH/F/98-, and TY/WI/66-like viruses. This study is a description of the previously uncharacterized H9N2 avian influenza viruses recently circulating in chickens and ducks in Mainland China. Our findings suggest that urgent attention should be paid to the control of H9N2 influenza viruses in animals and to the human's influenza pandemic preparedness.