The poultry  industry has become a critical component of the country's economy, and as a result, detecting and diagnosing disease infections before they spread in the herd limits the massive losses that poultry breeders face. This study was to look for serological diagnosis of mycoplasma disease in broiler using enzyme linked immune sorbent assay ELISA, as well as the histopathological changes in some organs. Between November 2021 and March 2022, this study was carried out broiler fields in Nineveh province. One hundred eighty blood samples were collected from 12 broiler fields. Results revealed the total infection rate of the examined fields was 55/180 (30.56%). In addition, percentage of M. gallisepticum infection was highest in the broiler fields located east of the province 21/45 (46.66%), followed by the percentage of infection in the fields located south of the province 18/45 (40%).  While the infection rate was lower in the fields on the north and west of the province, 11/45 (24.44%) and 5/45 (11.11%), While the infection rate was lower in the fields to the north and west of the province, 11/45 (24.44%) and 5/45 (11.11%), respectively.  Histopathological changes recorded severe tracheitis, severe inflammation in lung with purulent mucoid secretions in the bronchial and between the alveoli. The air sac showing also severe airsaculitis serous fibrin inflammatory secretion in the lumen with inflammatory cell of polymorphnuclear inflammatory cells infiltration and wall thickening. ELISA is a cost-effective tool for diagnosis of M. gallisepticum infection in broiler chickens, with high incidence in Nineveh province.