The emergence of highly pathogenic avian influenza (HPAI) H5N1 viruses and the risk of human pandemic have highlighted the need for advance stockpiling of vaccine. However, because of the rapid dissemination and ongoing evolution of avian H5N1 viruses, current vaccines may be sub-optimally matched to the actual pandemic virus. The authors reported here the evaluation of efficacy of NIBRG-14 vaccine (clade 1 A/Vietnam/1194/2004) against the H5N1 HPAl virus strains circulating in Vietnam. The birds were either vaccinated with single or booster dose of vaccine by subcutaneous injection then challenged with three H5N1 HPAI viruses (clade 1, clade 2.3.2.1 a and clade 2.3.2.1 b) at day 21st post-vaccination (p. v.). The results showed that NIBRG-14 protected birds from clade 1 and clade 2.3.2.1a infections. Notably, the authors observed that or booster dose of vaccine by subcutaneous injection then challenged with three H5N1 HPAI viruses (clade 1, clade 2.3.2.1 a and clade 2.3.2.1 b) at day 21 post-vaccination (p. v.). The results showed that NIBRG-14 protected birds from clade 1 and clade 2.3.2.1a infections. Notably, we observed that NIGRG-14 vaccine did not confer protection against clade 2.3.2.1b challenge virus. To get new insights of how H5N1 clade 2.3.2.1b (AiDuck/QuangNgai/1037/2011) virus can escape from host immune response induced by the vaccine, the study further analyzed the HA gene - a key virulence determinant of the virus. The result of amino acid sequence analyses indicated that this virus contained the sequence SPQRERRRK-R/G at the cleavage site in the HA molecule, indicating its high virulence. Additionally, we identified numerous mutations with amino acid substitutions in the hemagglutinin: M2261, 12398 located at N-link glycosylation site and 2H, 45N, 53K 1200, 133A and 14N mutations at antigenic site, which can affect receptor specificity as well as viral pathogenicity. Notably, 12398 and 8133A mutations are unique to AIDuck/QuangNgai/1037/2011, suggesting that it may involve in the virus ability to evade the host immune system. Taken together, phylogenetic analysis showed that continual mutations to the HA gene may generated novel antigenic strain and that probably changed the virulence of the virus and made the H5N 1 clade 2.3.2.1 b resistant to N IBRG-14 vaccine.