International audience<i>Mycoplasma synoviae</i> has been associated with economic loss in chickens and turkeys. The molecular characterization of <i>M. synoviae</i> at strain level allows the analysis of relationships between strains which may be valuable in epidemiological investigations. In this study, the intergenic spacer region (ISR) between the 16S and 23S rRNA genes was examined to see if useful information about strains could be derived. <i>M. synoviae</i> has two copies of this region, which may not be exactly the same (intercistronic heterogeneity). Sequencing of the ISRs of 21 <i>M. synoviae</i> isolates and the type strain revealed that 19 of them had such heterogeneity so DNA cloning was performed where necessary. All sequences were analysed and aligned; the percentage similarity of the DNA was calculated and a dendrogram was constructed. The length of the ISRs varied between 305 and 309 bp. Apart from having extra A/Ts in poly A or poly T regions and the presence of a few polymorphisms, the sequences of the <i>M. synoviae</i> strains were similar. Based on phylogenetic analysis the strains were assigned to 10 groups, taking into account that within each group the DNA similarity was 100%, while the lowest similarity between groups was 95.8%. The results were compared to those obtained with the <i>vlhA</i> gene, resulting in very similar <i>M. synoviae</i> groups. Although the ISR could be a good target for strain typing, as has been shown by others for <i>Mycoplasma gallisepticum</i>, the method may be too cumbersome for routine use with <i>M. synoviae</i> because of complications with intercistronic heterogeneity. However, if the ISR sequence information were to be combined with other mutation detection techniques it could increase the discriminatory power
