Mycoplasma hyospnoviae strains from Denmark, Germany, Japan, Sweden, the Netherlands and the UK were examined for variations in the genomic DNA and within the 16S ribosomal RNA (rRNA) gene. Variations in the chromosomal DNA among 57 isolates recovered from the respiratory tract and joints of pigs, were investigated by analysis of amplified fragment length polymorphisms of the Bg/II and MfeI restriction sites and by pulsed-field gel electrophoresis of a BssHII digest of chromosomal DNA. Both methods allowed unambiguous differentiation of the analysed strains and showed similar discriminatory potential for the differentiation of M. hyosynoviae isolates. Concordant results obtained with the two whole-genome fingerprinting techniques evidence the considerable intraspecies genetic heterogeneity of M. hyosynoviae. Sixteen field strains of M. hyosynoviae and the type strain S16(T) were further examined for variation within the 16S rRNA gene. Ten field strains possessed the 16S rDNA sequences identical to the type strain, while the remaining six strains had sequences that differed by one to two nucleotides from that obtained from the type strain.