Shahid and co-workers [7,13] discovered two specific SNPs on the obg gene and developed HRM-curve analysis based assays to differentiate the ts+ MS-H vaccine strain, ts- MS-H re-isolates and wild-type M. synoviae strains. In the current study we presented novel genotyping assays targeting these two SNPs on the obg gene which clearly differentiate the ts+ MS-H vaccine strain, ts- MS-H re-isolates and wild-type M. synoviae strains. Our assays are specific and sensitive enough (103−104 copy numbers) to detect and describe M. synoviae strains, when wild or vaccine strains are present as single specific pathogen in the sample. We also think that our assays are preferable to the assays developed earlier for the following three reasons: they are rapid, they can be performed directly on clinical samples (e.g. swabs) and the assays can be run simultaneously at the same annealing temperature. They are also simple as they can be performed on basic real-time PCR machines (without the HRM-curve analysis function) and on conventional PCR equipment coupled with agarose gel electrophoresis. They are cost effective as they do not require the expensive culture process, PCR product sequencing or costly reagents (e.g. TaqMan probes).