Fifteen strains of mAb specific for HA of the A/Hong Kong/482/97 (H5N1) influenza virus were generated. The HA antigenic sites of the human A/Hong Kong/482/97 (H5N1) influenza virus were defined by using yeast cell surface-displaying system and anti-H5 HA mAb. Evolution analysis of H5 HA identified residues that exhibit diversifying selection in the antigenic sites and demonstrated surprising differences between residue variation of H5 HA and H3 HA. A conserved neutralizing epitope in the H5 HA protein recognized by mAb H5M9 was found using viruses isolated from 1997-2006. Seven single amino acid substitutions were introduced into the HA antigenic sites, respectively, and the alteration of antigenicity was assessed. The structure obtained by homology-modeling and molecular dynamic methods showed that a subtle substitution at residue 124 propagates throughout its nearby loop (152-159). We discuss how the structural changes caused by point mutation might explain the altered antigenicity of the HA protein. The results demonstrate the existence of immunodominant positions in the H5 HA protein, alteration of these residues might improve the immunogenicity of vaccine strains.