An unusually high incidence of infectious keratoconjunctivitis followed by pneumonia and arthritis was observed in beef calves of a managed herd. No Moraxella spp. or bacteria other than Mycoplasma spp. were obtained from conjunctival and nasal swabs. A strategy was designed for characterization of bovine mycoplasmas at species and strain level on the basis of a combination of molecular tools and the immunoblotting method. The strategy made it possible to rapidly assign the bacterium responsible for this outbreak to one of the phylogenetic clusters of bovine mycoplasmas delineated in this study and then to identify it as Mycoplasma bovis. The strain, designated Sar 1, showed a 100% 16S rDNA sequence identity with two European strains (120/81 and MC3386) isolated in Germany and Ireland, respectively, and hosts a vsp gene analog to the vspA, vsp422-4, and vsp422-8 genes of the M. bovis reference strain PG45T and of the field strain 422. The use of a cross-reactive rabbit serum developed against the Mycoplasma agalactiae immunodominant antigen P48 confirmed the molecular findings. The immunological response of calves against M. bovis was also investigated. This is the first report on the occurrence of M. bovis on the Island of Sardinia (Italy).