Different methods were used for diagnosis of mycoplasma infection in layers farm at different ages. A total of one hundred and sixty four samples were collected as follow: (30) nasal swabs, 84 pharyngeal swab, 50, lung, air sac &trachea, and 96 serum samples. The primary isolation and identification revealed that 60 isolates Mycoplasma (M), from total of 164 organs samples (25/50isolates from lung, trachea and air sac (50%) at 3weeks of age, and 5/34 from pharyngeal swabs (14.7%), and 3/15 from nasal swabs (20%) at 13 weeks of age. Also MG isolates20/50 from pharyngeal swabs (40%), and 7/15 from nasal swabs (46.7) at 30 weeks of age. Antigenic and genetic variability between MG field isolates and reference MG strain (R) were studied by using polymerase chain reaction test (PCR) with primers complementary to the 16s rRNAgens was used to diagnose Mycoplasma gallisepticum field and reference strains which amplified by the oligonucleotide primers and gave acharacteristic fragment of 330bp. Diagnosis of MG by using molecular techniques is more specific and more rapid than conventional procedure. Also RAPD-PCR test could successfully differentiate between different MG strains. We concluded that individual strains of MG are genetically quite unique and this test is reliable method for diagnosis and differentiation of Mycoplasma gallisepticum Strains and can play an important role in understanding the epidemiology and spread of the disease, Serologic tests as Serum plate agglutination (SPA) and ELISA can indicate seroconversion; however, they could be used more satisfactorily for flock screening. Seven antibiotics were studied for their protection and treatment of mycoplasma ‘infection in laying birds. It was better to use norfloxacin, enrofloxacin, ciprofloxacin & Erythromycin to control MG.