The aim of this work was planned to study the prevalence of mycoplasma organisms in chickens from different localities of Kaluobia, Monofia and Gharbia governorates. In this study a total of 36 farms were examined for prevalence of mycoplasma organisms, these farms were : 3 layer, 20 broilers, 12 balady and one breeder, these farms were located in El Monofia, El Gharbia and El Kaluobia Governorates through application of two methods for diagnosis of Mycoplasma gallisepticum(MG) and Mycoplasma synoviae (MS). Flocks were examined for the detection of MG and MS infection by isolation and polymerase chain reaction (PCR). In layers 3 flocks were examined for presence of MG, the overall incidence were 14.66% out of the 12% from diseased birds and 2% from apparently healthy one. In broiler from 20 flocks were examined for presence of MG, the overall incidence were 30.38% out of the 33.63% from diseased birds and 12.5 % from apparently healthy. In balady from 12 flocks were examined for presence of MG, the overall incidence were 23.47% out of the 24.54% from diseased birds and 0 % from apparently healthy 4 out of 100 (4%) and 20 out 100 (20 %) for MS and MG respectively in diseased breeder in one flock. Mycoplasma gallisepticum field strain was sequenced and compared with the data base on Genbank. The Sequence analysis confirmed the presence of mgc2 virulent gene. The sequenced MG field strain was used in a laboratory experiment to confirm its pathogenicity through studying the clinical signs, body weight and histopathological lesions and minimum inhibitory concentration (MIC) for antibiotics and found that Tiamulin and Doxycycline gave lower concentration. Therefore, identification of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) using PCR is more accurate and faster than ordinary identification.