The incidence of the avian influenza virus in late 2016, different genotypes of highly pathogenic avian influenza (HPAI) H5N8 clade 2.3.4.4b have been reported among different domestic and wild bird species. The virus became endemic in the poultry population, causing a considerable economic loss for the poultry industry. This study screened 5 ostrich farms suffering from respiratory signs and mortality rate of the avian influenza virus. A flock of 60-day-old ostriches with a mortality of 90% suffered from depression, loss of appetite, dropped production, and oculo-nasal discharges, with bleeding from natural orifices as a vent. This flock was found positive for avian influenza virus and subtypes as HPAI H5N8 virus. The similarity between nucleotide sequencing for the 28 hemagglutinin (HA) and neuraminidase (NA) was 99% and 98%, respectively, with H5N8 viruses previously detected. The PB2 encoding protein harbor a unique substitution in mammalian marker 627A, which has not been recorded before in previously sequenced H5N8 viruses. Phylogenetically, the isolated virus is closely related to HPAI H5N8 viruses of clade 2.3.4.4b. The detection of the HPAI H5N8 virus in ostrich is highly the need for continuous epidemiological and molecular monitoring of influenza virus spread in other bird species, not only chickens. Ostrich should be included in the annual SunAlliance, for the detection of avian influenza.