This study evaluates the distribution of 20 variable regions in the genomes of 100 strains of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, and Mycobacterium bovis. The analysis reveals that most polymorphisms did not occur independently but resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis-specific deletion (TbD1), strains are divided into ancestral and "modern" groups, with the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African clusters. Successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum, M. microti, and M. bovis, which diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the hypothesis that M. tuberculosis evolved from M. bovis. M. canettii and ancestral M. tuberculosis strains lack these deleted regions, suggesting they are direct descendants of tubercle bacilli that existed before the M. africanum-M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli may have resembled M. tuberculosis or M. canettii and could have been a human pathogen. The study provides a new evolutionary scenario for the M. tuberculosis complex and highlights the importance of these variable regions as evolutionary markers and potential diagnostic tools.This study evaluates the distribution of 20 variable regions in the genomes of 100 strains of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, and Mycobacterium bovis. The analysis reveals that most polymorphisms did not occur independently but resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis-specific deletion (TbD1), strains are divided into ancestral and "modern" groups, with the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African clusters. Successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum, M. microti, and M. bovis, which diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the hypothesis that M. tuberculosis evolved from M. bovis. M. canettii and ancestral M. tuberculosis strains lack these deleted regions, suggesting they are direct descendants of tubercle bacilli that existed before the M. africanum-M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli may have resembled M. tuberculosis or M. canettii and could have been a human pathogen. The study provides a new evolutionary scenario for the M. tuberculosis complex and highlights the importance of these variable regions as evolutionary markers and potential diagnostic tools.