High pathogenicity avian influenza H5N1 has become an endemic poultry disease in several Asian countries, including Vietnam. Recently, clade 7 H5N1 viruses of the Eurasian lineage were isolated from chickens seized at ports of entry in Lang Son Province, Vietnam. Extensive nucleotide and amino acid divergence across the hemagglutinin (HA) protein gene of these isolates in comparison to previously described clade 7 viruses was identified. Clade 7 viruses are antigenically distinct from contemporary strains of H5N1 known to circulate in Vietnamese poultry (clade 1 and clade 2.3.4). Subsequent surveillance of sick poultry in live poultry markets in Hai Duong Province identified additional clade 7 isolates with HA genes very similar to the group B virus cluster detected previously at the Lang Son Province border. Antigenic analysis of the isolates from the live bird markets revealed significant cross-reactivity only between those clade 7 viruses belonging to the same subgroups. To meet pandemic response preparedness objectives, we have developed a reassortant virus from A/chicken/Vietnam/NCVD-016/2008, which could be used as a new prepandemic vaccine candidate for veterinary or human vaccination, should the need arise. Findings from these studies indicate that viruses with clade 7 HA have continued to evolve in Southeast Asian poultry, leading to significant antigenic drift relative to other H5N1 viruses currently circulating in Vietnam.