Mycoplasma gallisepticum (M.G) and Mycoplasma synoviae (M.S) have recognized as common respiratory pathogens especially in chickens cause a lots of economic losses in poultry industries. The aim of this study was development and validation of duplex Polymerase Chain Reaction (PCR) for simultaneous detection of MG and MS. A total of 50 samples from tracheas, lungs and air sacs were taken from commercial broiler chicken farms in Iran. The samples were cultured in PPLO broth supplemented for M.S and M.G isolation and bacteria DNA were extracted by phenol/chloroform. The conserved region of 16S rRNA gene was applied for the detection of Mycoplasma genus in 163bp fragment and MG in 183 bp fragment and vlhA gene for detection of MS in 350 bp fragment. Then duplex PCR amplified the conserved region of 16S rRNA and vlhA genes was applied for detection of MG and MS. 20 samples in Mycoplasma genus, 7 samples in MG and MS were positive in the single PCR whereas 3 samples simultaneously MG and MS were positive in the duplex PCR method. The results showed that duplex PCR was successful to simultaneous identification of MG and MS and suggested that duplex PCR is more rapid and inexpensive method than the single PCR for detection of MG and MS.