One key issue that needs to be analyzed for the above strategy is the cross reactivity between DNA vaccines expressing H5 HA antigens from different clades. It is critical to first optimize the immunogenicity of H5 HA DNA vaccines and then to test how much cross protection can be achieved with optimized H5 HA DNA vaccines. In the current report, we constructed DNA vaccines to express wild type HA antigens without mutations at the HA1 and HA2 cleavage site from four key H5N1 strains that have caused major human infection: HK/156/97 (clade 0), VN/1203/ 04 (clade 1), Ind/5/05 (clade 2.1), and Anhui/1/05 (clade 2.3). Rabbit sera immunized with these HA antigens were examined for their protective antibody responses against either homologous or heterologous H5N1 viruses. Our results demonstrated an imperfect cross-reactivity profile for the protective antibody responses among these four viruses. A polyvalent formulation including three different H5 HA DNA vaccines was able to produce broad protective antibody responses with high titers against these key H5N1 isolates. Information learned from this study should facilitate the selection of candidate H5N1 vaccines to form polyvalent H5N1 DNA vaccines as part of the global strategy to prevent and control a potential avian flu pandemic.