Abstract: The study was aimed at establishing a triplex PCR to differentiate M.bovis, M. mycoides subsp. mycoides Small Colony Type and M. agalactiae out of the same sample, for clinical diagnosis and epidemiological survey. Three pairs of primers were designed and synthesized according to the published literatures in GenBank. And a triple PCR was developed to amplify conservative regions of each bacterial genomes of M. Bovis, M. mycoides subsp- mycoides Small Colony Type and M. agalactiae, with the target sequences of 448, 549 and 375 bp, respectively. Sensitivity of this triple PCR was analyzed. Specificity was verified with template from M. gallisepticum, M. Swine, M. agalactiae and M. mycoides subsp- mycoides Small Colony. And reliability was tested by comparing the results from determinative bacteriology. Three specific fragments of 448, 549 and 375 bp, corresponding to M. Bovis, M. mycoides subsp- mycoides Small Colony Type and M. agalactiae, were amplified under optimized condition, without interference between templates and the primers. And results were in agreement with determinative bacteriology. No band was shown in targeting genomes from M. gallisepticum and M. Swine. Sensitivity was determined as 0.8 ng·μL^-1 These results suggest that the triplex PCR could differentiate M. Bovis, M. mycoides subsp- mycoides Small Colony Type and M. agalactiae based on one single sample, with high sensitivity, specificity and reliability，and can be applied in clinical diagnosis and epidemiological survey.