This study investigates the phenomenon of IS6110-mediated deletion polymorphism in the direct repeat (DR) region of the genome of Mycobacterium tuberculosis. Clinical isolates and their putative predecessors were compared using a combination of DR region restriction fragment length polymorphism, IS6110 DNA fingerprinting, spoligotyping, and DNA sequencing, which allowed the mapping of chromosome structure and deletion junctions. The data suggest that adjacently situated IS6110 elements mediate genome deletion. However, in contrast to previous reports, deletions appear to be mediated by inversely oriented IS6110 elements. This suggests that these events may occur via mechanisms other than RecA-mediated homologous recombination. The results underscore the important role of IS6110-associated deletion hypervariability in driving M. tuberculosis genome evolution.