An inactivated H5N1 avian influenza (AI) vaccine generated by reverse genetics and containing the hemagglutinin and neuraminidase genes of the H5N1 A/goose/Guangdong/1/96 (GS/GD/06) virus has been used in domestic poultry in China and Vietnam as an important control strategy for H5N1 AI. The efficacy of this vaccine against early H5N1 isolates has been fully evaluated in chicken, ducks, and geese. However, there are no reports about its efficacy against H5N1 viruses recently isolated in China and Vietnam. In this study, groups of 3-wk-old specific-pathogen-free chickens were intramuscularly injected with one dose of the vaccine. Three weeks postvaccination, the chickens were intranasally challenged with 10(5)EID50 of six different lethal H5N1 AI viruses: A/bar-headed goose/Qinghai/3/05 (clade 2.2), A/chicken/Shanxi/2/06 (CK/SX/06; clade 7), A/duck/Fujian/31/07 (clade 2.3.4), A/MDK/VN-HD/46/07 (clade 2.3.4), A/MDK/VN-CM/1185/06 (clade 1), and A/MDK/VN-CM/1159/06 (clade 1). Four out of 20 chickens challenged with the CK/SX/06 shed virus on day 5 and died on day 8 to 9 postchallenge. Chickens challenged with the remaining five viruses were completely protected (no disease signs, virus shedding, or deaths). These results indicate that the GS/GD/06-based vaccine provides sound protection against clade 1, 2.2, and 2.3.4 viruses, but not against the CK/SX/06 virus, which emerged in northern China in 2006.