One-year surveillance of HPAI (H5N1) virus infection in different chicken flocks (141 flock) from different Governorates in Upper Egypt was carried out, during the period from January 2015 to January 2016.  The detection rate of highly pathogenic avian influenza (HPAI) H5N1 virus was 21.3% (30/141) which was highest during the winter season compared with the other seasons. The investigated flocks suffered from respiratory and nervous signs and cyanosis of the comb and wattles was observed in some birds as well as subcutaneous hemorrhages in shanks. The thirty positive H5N1 samples were tested for the existence of other respiratory viruses. The results were as follows: single H5N1 virus infection was only detected in 5 flocks (16.6%). The mixed H5N1 with H9N2 in 2 flocks (6.6%); H5N1with NDV in 12 farms (40%); H5N1 with IBV in 7 farms (23.3%) while H5N1, H9N2 and NDV were detected together in 4 flocks (13.3 %). The sequence analysis of the HA gene from five selected H5N1 isolates revealed that these strains were clustered with Egyptian classical H5N1 viruses (sub-clade 2.2.1.2) of the Eurasian origin. It is concluded that, the five H5N1 subtype isolates of HPAI are clustered with classical H5N1 viruses to subclade 2.2.1.2 of Eurasian origin. Therefore, vaccination of the backyard chicken as well as chicken farms with suitable homologues vaccine to eliminate HPAI virus from Egypt is recommended.