The evolution of the major surface hemagglutinin (HA) antigen of type A H5N1 influenza viruses is explored at the amino acid level using a new proteotyping approach. Alignments of translated hemagglutinin gene sequences of all characterised type A H5N1 strains, or subsets thereof, has enabled the presence of signature peptides of conserved sequence and unique mass to be investigated from the perspective of the host, period and region from which strains were isolated. Consistent with the rapid, cross species transmission of H5N1 strains among migratory birds, poultry and humans throughout south-east Asia, no signatures unique to the host or region were found. Nevertheless, several period-specific signature peptides were identified that enable strains associated with the 1997 H5N1 pandemic to be rapidly differentiated from those in circulation across the subsequent decade.