Abstract: Fifty three of Middle East avian influenza virus H5N1 strains were retrieved from GenBank Database. Other 23 H5N1 representative strains from Asian, Europe, Africa, and North America were also used in this study. Phylogenetic analysis and pairwise comparison of nucleotide and amino acid sequences of.hemagglutinin (HA) gene region of these strains revealed that the H5N1 viruses circulating in Middle East displayed high similarity. The HA protein of the virus contained multiple basic amino acid residues (QGERRRKKR) or (QGEGRRKKR) adjacent to the cleavage site between the HA1 and HA2 domains, showing the highly pathogenic characteristics. Further analyses of these H5N1 strains showed that all of them carried (Gln) 238Q and (Gly) 240G at the receptor binding pocket which indicates preferential binding to alpha-2,3-NeuAcGal receptors. Phylogenetic analysis also confirmed that these H5N1 viruses belonged to the Qinghai lineage or Astrakhan lineage of highly pathogenic avian influenza (HPAI) viruses H5N1 (subclade 2.2 or EMA). As a result, 2 closely related but distinguishable H5N1 sub-subclades are defined and called (2.2.1 (EMA-1) and 2.2.3 (EMA3).