We performed an in-depth analysis of a large Mycobacterium tuberculosis cluster involving nine isolates with identical or highly similar RFLP types. The strain involved in this transmission cluster microevolved and accumulated up to five differential IS6110 transposition events, several of them not revealed by standard genotyping approaches. Some of these events probably generated extended deletions. In two cases of tuberculosis within the cluster, we observed the simultaneous presence of both the reference and microevolved variants. A longitudinal genotypical survey of M. tuberculosis in a real clinical evolutionary scenario can reveal dynamic and complex microevolution events.