Mycoplasma Synoviae (MS) is one the major pathogens of avian, causing significant economic losses of poultry industry. We examined the differentiating potential of diagnostic polymerase chain reaction (PCR) primers targeted to the gene VIhA present in MS. For serological screaming test, we selected 10 farms and taking blood samples for RSAT assay. For VlhA-PCR assay, we were taking 10 samples included: lung, air sacs and tracheal swabs. The VIhA-PCR diagnostic primers are specific for MS in tests of all avian mycoplasmas or bacteria present in the chicken trachea and are sensitive enough to readily detect MS in tracheal swabs from field outbreaks. 350-400 bp PCR products on electrophoresis gel appears with VIhA-PCR diagnostic primers are specific for MS. The test was successfully applied in vivo for detection of MS in clinical samples.