Mycoplasma synoviae (MS) is a poultry pathogen that has had an increasing incidence and economic impact over the past few years. Strain identification is necessary for outbreak investigation, infection source identification and facilitating prevention and control and eradication efforts. Currently, a segment of the variable Lipoprotein Hemagglutinin A (vlhA) gene (420 bps) is the only target that is used for MS strain identification. A major limitation of this assay is that colonality of typed samples can only be inferred if their vlhA sequences are identical; however, if their sequences are different, the degree of relatedness is uncertain. In this study we propose a multilocus sequence typing (MLST) assay to further refine MS strain identification. After initial screening of 24 housekeeping genes as potential targets, 7 genes were selected for the MLST assay. An internal segment (450 bps - 711 pb) from each of the 7 genes was successfully amplified and sequenced from 58 different MS strains and field isolates (N=30) or positive clinical samples (N=28). The collective sequence of all 7 gene segments (3960 bps total) was used for MS sequence typing. The 58 tested MS samples were typed into 30 different sequence types using the MLST assay, and coincidentally all the samples were typed into 30 sequence types using vlhA assay. However, the phylogenetic tree generated using the MLST data was more congruent to the epidemiological information than the tree generated by vlhA assay. We suggest that the newly developed MLST assay and the vlhA assay could be used in tandem for MS typing. The MLST assay will be a valuable and more reliable tool for MS sequence typing, providing better understanding of the epidemiology of MS infection. This in turn will aid disease prevention, control and eradication efforts.