This study aimed to molecular diagnose hydropericardium hepatitis syndrome (HHS) in the broiler chickens of six areas in Nineveh Province, Iraq namely: Mosul, Talafer, Baaj,  Bartela, Hamdanya and Qayara during the period October 2021-March 2022. Clinical and pathological manifestation of the disease in affected birds was investigated and observed. A total of 24 infected liver lesioned samples as 12 broiler farms (2 farms for each area) detected for hexon gene of FAdV by PCR and sequencing for accurate diagnosis. The results revealed that the PCR of liver samples for 12 broiler farms of the six regions in Nineveh Province were positive results of HHS adenovirus 897bp and the result of DNA sequencing revealed that the pathogenic HHS adenovirus of the six areas was serotype 4 of one strain E1B corresponding with sequencing XM_040669733.2 of National Center for Biotechnology Information NCBI. This study is the first of its kind for the molecular diagnosis and determination of the strain type of FAdV that causes HHS in broilers chicken in Nineveh Province, Iraq.