The spread of H5N1 virus into a wide range of avian and mammalian species may facilitate the adaptation of the virus to human populations. In the present study, a survey of avian influenza virus in tree sparrows was performed and a HPAI H5N1 virus (A/tree sparrow/Jiangsu/1/08) was isolated. The H5N1 virus was found to be a genotype V variant belonging to clade 2.3.4, which had newly emerged in southern China in 2005. Genetic analysis showed that it had a close relation with A/Jiangsu/1/07 (H5N1), which was thought to be responsible for a probable limited person-to-person transmission in a family cluster in China in 2007. Pathogencity studies showed that the virus was highly virulent when experimentally inoculated into the chickens, tree sparrow, and mouse. As clade 2.3.4, genotype V variant of H5N1 virus has been accounted for the most of human fatal cases in China during 2005-2008, the existence of such H5N1 variants in the tree sparrows highlights the potential threat of this type of wild bird infection to veterinary and public health.