AIM: To develop real-time PCR assays for the detection and differentiation of members of the Mycoplasma mycoides cluster. METHODS: Five real-time PCR assays were designed to al-low differentiation of members of the M mycoides cluster: an assay for detection of the M mycoides subspecies, viz M mycoides subsp mycoides large colony (MmmLC), M mycoides subsp capri (Mmc), and M mycoides subsp mycoides small colony (MmmSC); one for the detection of the M capricolum subspecies, viz M capricolum subsp capricolum (Mcc), M capricolum subsp capripneumoniae (Mccp), and Mycoplasma sp bovine group 7 (BG7); and three for the specific detection of MmmSC, Mccp, and BG7. A panel of 74 Mycoplasma isolates from various geographical origins and a panel of 21 other bacterial isolates were used to evaluate the sensitivity and specificity of the as-says. RESULTS: The assays displayed 100% analytical sensitivity in detecting all target Mycoplasma isolates. The analytical detection limit for the assays to detect the M mycoides subspecies, M capricolum subspecies, and MmmSC was determined to be 100 fg of genomic DNA, while the Mccp and BG7 assays had a detection limit of 100 fg and 10 fg of genomic DNA, respectively. The M mycoides subspecies assay had a detection limit of 103 (SD 102) cfu/ml milk, 104 (SD 104) cfu per swab, and 103 (SD 103) cfu/g lung in inoculated samples. The assays displayed 100% specificity when applied to non-target bacterial isolates and to 110 culture-negative milk samples. CONCLUSIONS: The assays were highly sensitive and specific, and provide accurate detection and differentiation of the members of the M mycoides cluster.