H5N1 avian influenza virus has caused widespread infection in poultry and wild birds, and has the potential to emerge as a pandemic threat to humans. The hemagglutinin (HA) is a glycoprotein on the surface of the virus envelope. Understanding its antigenic structure is essential for designing novel vaccines that can inhibit virus infection. The aim of this study was to map the amino acid substitutions that resulted in resistance to neutralization by monoclonal antibodies (MAbs) of the highly pathogenic A/crested eagle/Belgium/01/2004 (H5N1), a clade 1 virus. Two hybridomas specific to H5N1 clade 1 viruses were selected by enzyme-linked immunosorbent assay, virus neutralization test, and immunofluorescence assay. Escape mutant populations resisting neutralization by those MAbs (8C5 and 5A1) were then selected, and sequencing of these mutants allowed the prediction of the HA protein structure by molecular homology. We could detect an amino acid change in our escape mutants at position K189E corresponding to antigenic site 2 of H5 HA1 and site B of H3 HA1. Interestingly, 336 out of 350 available HA sequences from H5N1 clade 1 and clade 2.3 viruses had Lys (K) at position 189 in the HA1, whereas HA sequences analyzed from clade 2.1 and 2.2 viruses had Arg (R). This residue also interacts with the receptor-binding site, and it is thus important for the evolution of H5N1 viruses. An additional substitution K29E in HA2 subunit was also observed and identified with the use of NetChop software as a loss of a proteasomal cleavage site, which seems to be an advantage for H5N1 viruses.