This study aimed to optimize a method based on the polymerase chain reaction - multiplex PCR - for differentiation of mycobacteria species of interest for public health. The multiplex PCR was based on simultaneous amplification of the hsp65 gene, which is present in all species of the Mycobacterium genus, the dnaJ gene, which is present only in Mycobacterium tuberculosis and Mycobacterium avium and the IS6110 insertion sequence, which is present in the Mycobacterium tuberculosis complex, generating amplicons of 165 bp, 365 bp and 541 bp, respectively. The detection limit was 1 fg for the hsp65 target, 100 pg for dnaJ and 0.1 fg for IS6110. The multiplex PCR detected down to 100 pg of DNA of Mycobacterium tuberculosis. The system was shown to be specific and sensitive for detection of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. The results obtained using reference strains of mycobacteria showed that multiplex PCR may be a fast, sensitive and specific tool for differentiation of mycobacteria.