A new evolutionary scenario for the Mycobacterium tuberculosis complex has been proposed based on the analysis of 20 variable genomic regions in 100 strains of the M. tuberculosis complex, including M. tuberculosis, M. africanum, M. canettii, M. microti, and M. bovis. These regions, which result from insertion-deletion events, were found to be shared among ancestral strains rather than occurring independently in different strains. This suggests that the majority of these polymorphisms originated from ancient genetic events in common progenitor strains. The presence or absence of a specific deletion (TbD1) in M. tuberculosis strains divides them into ancestral and "modern" strains, with the latter representing major epidemic clusters such as Beijing, Haarlem, and African strains. Successive loss of DNA, reflected by regions of difference (RDs), was identified in lineages that diverged from the progenitor of M. tuberculosis before TbD1 occurred. These findings contradict the hypothesis that M. tuberculosis evolved from M. bovis. Instead, 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 implies that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could have been a human pathogen. The M. tuberculosis complex includes species that are closely related, with 99.9% nucleotide similarity and identical 16S rRNA sequences, but differ in host tropism, phenotypes, and pathogenicity. Some are exclusively human pathogens, while others have a wide host range. The study reveals that the genetic organization of the last common ancestor of the tubercle bacilli and its host is still unclear. The evolutionary bottleneck hypothesis, suggesting that the M. tuberculosis complex underwent a bottleneck 15,000–20,000 years ago, is supported by the high conservation of housekeeping genes. The study also shows that the TbD1 deletion is a key marker for distinguishing modern M. tuberculosis strains from ancestral ones. The analysis of RD and TbD1 regions in various strains provides insights into the evolutionary history of the complex, showing that M. canettii and ancestral M. tuberculosis strains are closely related to the common ancestor. The findings suggest that the M. tuberculosis complex evolved from a common ancestor that was already a human pathogen, and that the TbD1 deletion occurred in a lineage that diverged from the M. tuberculosis lineage. The study highlights the importance of deletion analysis and molecular typing in understanding the evolution and identification of the M. tuberculosis complex.A new evolutionary scenario for the Mycobacterium tuberculosis complex has been proposed based on the analysis of 20 variable genomic regions in 100 strains of the M. tuberculosis complex, including M. tuberculosis, M. africanum, M. canettii, M. microti, and M. bovis. These regions, which result from insertion-deletion events, were found to be shared among ancestral strains rather than occurring independently in different strains. This suggests that the majority of these polymorphisms originated from ancient genetic events in common progenitor strains. The presence or absence of a specific deletion (TbD1) in M. tuberculosis strains divides them into ancestral and "modern" strains, with the latter representing major epidemic clusters such as Beijing, Haarlem, and African strains. Successive loss of DNA, reflected by regions of difference (RDs), was identified in lineages that diverged from the progenitor of M. tuberculosis before TbD1 occurred. These findings contradict the hypothesis that M. tuberculosis evolved from M. bovis. Instead, 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 implies that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could have been a human pathogen. The M. tuberculosis complex includes species that are closely related, with 99.9% nucleotide similarity and identical 16S rRNA sequences, but differ in host tropism, phenotypes, and pathogenicity. Some are exclusively human pathogens, while others have a wide host range. The study reveals that the genetic organization of the last common ancestor of the tubercle bacilli and its host is still unclear. The evolutionary bottleneck hypothesis, suggesting that the M. tuberculosis complex underwent a bottleneck 15,000–20,000 years ago, is supported by the high conservation of housekeeping genes. The study also shows that the TbD1 deletion is a key marker for distinguishing modern M. tuberculosis strains from ancestral ones. The analysis of RD and TbD1 regions in various strains provides insights into the evolutionary history of the complex, showing that M. canettii and ancestral M. tuberculosis strains are closely related to the common ancestor. The findings suggest that the M. tuberculosis complex evolved from a common ancestor that was already a human pathogen, and that the TbD1 deletion occurred in a lineage that diverged from the M. tuberculosis lineage. The study highlights the importance of deletion analysis and molecular typing in understanding the evolution and identification of the M. tuberculosis complex.