Abstract: To develop a rapid PCR assay for simultaneous detection of Mycoplasma gallisepticum (MG) and Mycoplasma synovial (MS), two pairs of primers were designed and synthesized according to the published sequences of the gapA gene of MG and the heat shock ATP-dependent protease gene of MS in GenBank, and a duplex PCR protocol for detection of MG and MS was developed by optimizing conditions of PCR reaction. The specific fragments of 729 bp for MG and 309 bp for MS were simultaneously amplified in the duplex PCR, but no PCR products were amplified for Pasteurella multocida,Escherichia coli,Samonella pullorum and Avibacterium paragallinarum. The detection limit was 5×10^-2 ng/μL for both of MG and MS. Detection results from clinical samples demonstrated that the co-infection of MG and MS as well as single infection of MG or MS could be found by using the established duplex PCR assay. In conclusion, the duplex PCR assay has good specificity, sensitivity and repeatability, which provides effective technical support for clinical diagnosis of MG and MS co-infection in veterinary clinic.