The MS-H live attenuated vaccine strain (Vaxsafe MS®; Bioproperties Pty. Ltd., Australia) is commonly used around the world to control chronic infections caused by Mycoplasma synoviae in birds and to minimise economic losses in the poultry industries. The MS-H strain is a temperature-sensitive (ts+) derivative of a virulent field strain 86079/7NS obtained by chemical mutagenesis (1-3). There are 32 single nucleotide polymorphisms (SNPs) in MS-H compared to 86079/7NS, from which 25 were detected in predicted coding regions (CDSs) (4).There is limited information available on the stability of these mutations in MS-H after vaccination of chickens (5-8). In this study, we sequenced the whole genome of fifty-five MS-H reisolates collected from vaccinated poultry flocks in nine countries, and compared these sequences to five commercial vaccine batches. We also sequenced the whole genome of the master and working seeds, which are used to produce the commercial MS-H vaccine. The genomes were aligned to the published MS-H genome sequence (Genbank accession number CP021129). The genomes of commercial vaccine batches were identical to those of master and working seed batches. However, the genomes of the MS-H re-isolates contained a total number of 490 variations, from which 314 occurred in 133 predicted CDSs. Of these CDS-associated variations, 89 were detected in in more than one genome, indicating these areas are mutation “hotspots. Of the 25 CDSs containing mutations previously found between 86079/7NS and MS-H, only 8 were found among the hotspots and the remaining 17 were considered not to be prone to any mutations or reversions. This study suggests that most of the mutations of MS-H strains are stable and therefore, the vaccine should be considered a safe live attenuated vaccine. Also, the stable mutations could be used for Differentiation of Infected from Vaccinated Animals (DIVA) methods to distinguish between the field and vaccine strains.