The highly pathogenic avian influenza (HPAI) H5N1 virus has received considerable attention during the past 2 decades due to its zoonotic and mutative features. This Virus is of special importance due to to the possibility of causing infection in human populations. According to it´s geographical location, Iran hosts a large number of aquatic migratory birds every year, and since these birds can be considered as the host of the H5 HPAI, the country is significantly at risk of this virus. the In this study, the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) genes of the H5N8 strain were identified in Malard county of Tehran province and Meighan wetland of Arak city, Markazi province were investigated. Based on the analysis of the amino acid sequence of the HA genes, the cleavage site of the gene includes the PLREKRRKR/GLF polybasic amino acid motif, which is a characteristic of highly pathogenic influenza viruses. The HA gene of two viruses had T156A, S123P, S133A mutations associated with the increased mammalian sialic acid-binding, and the NA gene of two viruses had H253Y mutations associated with the resistance to antiviral drugs. Phylogenetic analysis of the HA genes indicated the classification of these viruses in the 2.3.4.4 b subclade. Although the A/Goose/Iran/180/2016 virus was also an H5N8 2.3.4.4 b virus, its cluster was separated from the A/Chicken/Iran/162/2016 virus. This means that the entry of these viruses in to the country happened through more than one window. Furthermore, it seems that the introduction of these H5N8 HPAI strains in Iran probably occurred through the West Asia-East African flyway by wild migratory aquatic birds.