Parasitic mycobacteria cause important human and animal diseases including tuberculosis, leprosy, and paratuberculosis. Several methods demonstrate a high degree of sequence conservation in three parasitic mycobacterial species (Mycobacterium tuberculosis, M. leprae, and M. avium subspecies paratuberculosis). Each of these species has completely conserved deoxyribonucleic acid (DNA) sequence in an internal transcribed spacer. In contrast, several species of environmental mycobacteria (M. intracellulare, M. kansasii, M. gordonae, and M. scrofulaceum) have substantial strain-to-strain variation in this region. These data suggest that each of the parasitic species has gone through a recent evolutionary bottleneck. Comparisons of tandem-repeat DNA from ancient and modern mycobacterial strains may allow this hypothesis to be tested directly.