Fourteen Mycoplasma strains were isolated from the oral cavity and genital tract of asymptomatic dogs. Isolates had been preliminarily identified by conventional serological testing as Mycoplasma bovigenitalium, but in 16S-23S rRNA intergenic spacer PCR-RFLP assays the isolates exhibited an RFLP pattern distinct from M. bovigenitalium PG11(T). Analysis of the 16S rRNA gene placed a representative of the isolates (strain 1642(T)) in the M. bovigenitalium subcluster of the Mycoplasma bovis cluster of mycoplasmas, with the highest sequence similarities to Mycoplasma californicum ST-6(T) (96.4 %), M. bovigenitalium PG11(T) (96.3 %) and Mycoplasma phocirhinis 852(T) (96.2 %). 16S rRNA gene sequence similarities almost equidistant from three recognized species and results obtained by sequence analysis of the 16S-23S rRNA intergenic spacer region, polar lipid profiles and serological reactions indicated that this organism represents a novel species of the genus Mycoplasma for which the name Mycoplasma mucosicanis sp. nov. is proposed, with strain 1642(T) ( = ATCC BAA-1895(T) = DSM 22457(T)) as the type strain.