Mycoplasmas of the "Mycoplasma mycoides" cluster are all ruminant pathogens. Mycoplasma mycoides subsp. mycoides (MmmSC) is responsible for contagious bovine pleuropneumonia and is known to produce capsular polysaccharide (CPS) and exopolysaccharide (EPS). Previous studies have strongly suggested a role for MmmSC polysaccharides in pathogenicity. MmmSC-secreted EPS was recently characterized as a β-(1-6)- galactofuranose homopolymer (galactan) identical to the capsular product. Here, we extended the characterization of secreted polysaccharides to all other members of the "M. mycoides" cluster: M. capricolum subsp capripneumoniae (Mccp) M. capricolum subsp capricolum (Mcc), M. leachii and M. mycoides subsp capri (Mmc, including the "LC" and "capri" serovars). Extracted EPS were characterized by nuclear magnetic resonance, resulting in the identification of a homopolymer of β-(1-2)-glucopyranose (glucan) in Mccp and M. leachii. Monoclonal antibodies specific for this glucan and for the MmmSC-secreted galactan, were used to detect the two polysaccharides. While Mmc strains of the "LC" serovar produced only capsular galactan, no polysaccharide could be detected in strains of the "capri" serovar. All strains of Mccp and M. leachii produced glucan CPS and EPS, whereas glucan production and localization varied among Mcc strains. Genes associated with polysaccharide synthesis, and forming a biosynthetic pathway, were predicted in all cluster members. These genes were organized in clusters within two loci representing genetic variability hotspots. Phylogenetic analysis showed that some of these genes, notably galE and glf, were acquired via horizontal gene transfer. These findings call for a reassessment of the specificity of the serological tests based on mycoplasma polysaccharides.< Réduire