The assumption that Mycobacterium tuberculosis infections should be considered clonally homogeneous has been weakened in the last few years. Recent studies have shown (i) the isolation of different M. tuberculosis strains from sequential episodes, (ii) mixed infections by two M. tuberculosis strains, and (iii) genetic variations in M. tuberculosis subpopulations due to microevolution events. Nevertheless, it is unknown whether clonal heterogeneity could be found in the initial steps of M. tuberculosis infection, i.e., the primary infection. In the present study we analyzed the clonal composition of the M. tuberculosis isolates causing primary infections in children. Cultures were clonally homogeneous in most cases (11 of 12). In 1 of the 12 cases (8.3%), clonal heterogeneity among the M. tuberculosis isolates was found by spoligotyping and IS6110-based restriction fragment length polymorphism analysis. This case occurred in a 2-year-old child in whom microevolution events were unlikely and who had no risk factors for overexposure to M. tuberculosis. Clonal heterogeneity should also be considered in primary M. tuberculosis infections, including circumstances in which it is usually unexpected.