An inactivated H5N1 avian influenza (AI) vaccine (Re-6) that bears the HA and NA genes from a clade 2.3.2.1 H5N1 virus, A/duck/Guangdong/S1322/10 (DK/GD/S1322/10), has been used in domestic poultry in China and other Southeast Asian countries to control clade 2.3.2.1 H5N1viruses since 2012. The efficacy of this vaccine against H5N1 viruses isolated in recent years has not been reported. In this study, we evaluated the protection efficacy of the Re-6 vaccine in chickens against challenge with four clade 2.3.2.1 H5N1 viruses, one clade 2.3.4.4 H5N1 virus, and one clade 7.2 H5N1 virus; these viruses were isolated in mainland China, Hong Kong, and the Democratic People's Republic of Korea between 2011 and 2015. The vaccinated chickens were completely protected (no disease signs, virus shedding, or death) from the challenge with the four clade 2.3.2.1 H5N1 viruses. In the clade 7.2 virus–challenged group, all of the vaccinated chickens remained healthy and survived for the entire 2-wk observation period; virus shedding was only detected from 1 of 10 chickens on day 3 postchallenge. In the clade 2.3.4.4 virus–challenged group, 8 of the 10 vaccinated chickens remained healthy and survived the 2-wk observation period; however, virus shedding was detected from 8 of 10 chickens on day 5 postchallenge. These results indicate that the Re-6 vaccine provides solid protection against clade 2.3.2.1, good protection against clade 7.2, and poor protection against clade 2.3.4.4.