The avian influenza virus is a highly contagious viral disease that predominantly impacts avian species, especially domestic birds like chickens, ducks, and turkeys. The study was conducted to isolate the avian influenza virus from domestic poultry breeds (Desi, Aseel, Fayoumi, and Golden) from backyard poultry population of the Tehsil Abbottabad from January to June 2023. A cross-sectional study was conducted in three rural and five urban areas of Tehsil Abbottabad Using sterile cotton swabs, 128 (tracheal and cloacal) swab samples were collected. The samples were labelled and stored in a brain-heart infusion medium, then transported to the National Reference Laboratory for Poultry Diseases in Islamabad using a thermostat shipment box. The samples were processed using a real-time reverse transcription polymerase chain reaction for avian influenza virus detection. This study concludes with a significant 9.38% prevalence percentage of avian influenza virus in Tehsil Abbottabad backyard poultry. The prevalence percentage of H9 was 83.33%, and that of H5 was 16.66%. The findings reveal the importance of implementing preventative measures to curb avian influenza outbreaks within the poultry population of the district. This study offers vital data for AIV research in 2023, guiding enhanced containment and prevention in backyard poultry to control avian influenza.