We developed a novel multiplex PCR assay using dual-priming oligonucleotide primers targeting the RD1 gene for simultaneous identification of the Mycobacterium tuberculosis complex and M. bovis bacillus Calmette-Guérin (BCG). This assay would be useful both for detection of the M. tuberculosis complex and for differentiation of M. bovis BCG from pathogenic M. tuberculosis complex species.