Differentiation of members of the Mycobacterium tuberculosis complex by conventional mycobacteriological methods is time consuming, making surveillance of species-specific disease difficult. A two-step, multiplex polymerase chain reaction (PCR) method based on genomic regions of difference (RD1, RD1(mic), RD2(seal), RD4, RD9 and RD12) was developed for the differentiation of M. canettii, M. tuberculosis, M. africanum, M. microti, M. pinnipedii, M. caprae, M. bovis and M. bovis BCG. The size of the respective multiplex PCR amplification products corresponded to the presence of the different M. tuberculosis complex members. This method allows for rapid differentiation, making it suitable for routine laboratories and surveillance purposes.