The aim of this study was to identify molecular techniques which enable clear discrimination between Mycoplasma synoviae isolates for improved epidemiology. Multilocus sequence analysis (MLSA) of 6 M. synoviae loci was conducted for genotyping of isolates with previously determined 5'-vlhA sequences. Sequencing of three polymorphic genes (5'-vlhA, cysP and nanH) enables good discrimination between isolates with different genotypes. Such a genotyping scheme revealed 10 distinct genotypes, which were confirmed by sequencing of an additional three loci of the M. synoviae genome. Epidemiologically linked strains formed clusters with the same genotypes which clearly differed between clusters. MLSA used in this study is a promising tool for epidemiology of M. synoviae isolates, but it should be evaluated by further investigations using a much higher number of M. synoviae strains.