The entire genome of the A/Chicken/Hubei/C1/2007 (H9N2) virus, isolated from central China in 2007, was completely sequenced and phylogenetically analyzed. Phylogenetic analysis demonstrated that A/Chicken/Hubei/C1/2007 (H9N2) virus represents multiple reassortant lineages, with genes coming from the early mainland China strain (Ck/Bejing/1/94), an H9N2 virus with special genotype (Ck/shanghai/F/98) and other lineages from poultry in Asia. Infection studies indicated that A/Chicken/Hubei/C1/2007 (H9N2) virus replicated efficiently in MDCK cells and in BALB/c mice. The H9N2 virus also replicated to high titers in chicken respiratory tracts and caused overt clinical signs in chickens. Our results suggest that attention should be paid to the natural evolution of H9N2 influenza viruses and to the control of H9N2 influenza viruses in animals.