The Mycobacterium tuberculosis complex includes the four species M. tuberculosis, M. bovis, M. africanum, and M. microti. We sequenced 13 M. tuberculosis complex strains in the 16S-to-23S rDNA internal transcribed spacer (ITS). The ITS has a high rate of nucleotide substitution. Previous reports found three nucleotide substitutions in the ITS between two M. tuberculosis complex strains. In contrast, we found the same ITS sequence in all 13 M. tuberculosis complex strains (including all four species and M. bovis BCG). This finding confirms the conservation of 16S rDNA sequence and the high DNA-DNA relatedness found in previous studies. By the usual criteria, the four species of the M. tuberculosis complex would be considered a single species. In a phylogenetic analysis based on the ITS sequence, the four species of the M. tuberculosis complex were distinct from nontuberculous mycobacteria. The ITS contains at least seven potential sites for oligonucleotide probes with specificity for the M. tuberculosis complex.