Mycoplasma synoviae, (MS) as a pathogen involving in respiratory and locomotor disorders, causes many economic losses on poultry industry in Iran. Therefore, early and reliable diagnosis and also strain differentiation is a key to prevention and control. The aim of this study was the isolation, detection, and differentiation of Iranian isolates especially field and vaccine strains using PCR. Eleven serologically positive flocks from different parts of country were selected. From each flock, 20 blood samples were provided for serum plate agglutination test and ELISA; 10 swabs were taken from trachea, cleft palate, and tissues for MS isolation; and 9 swabs were collected for PCR identification. For the first time in Iran, the primers complementary to the single-copy conserved 5' end of vlhA gene were used for detection of MS by PCR. Results obtained from serology, isolation, and PCR using primers related to 16s rRNA and vlhA genes were analyzed and compared. PCR results, in addition to identification of Mycoplasma species, revealed variable sizes of 350-400 bp among standard strain, vaccine strains, and Iranian field isolates. The findings of this study demonstrated that the vlhA gene-targeted PCR is a sensitive and specific test for detection of M. synoviae, and an efficient tool for primary typing of its different strains.