An optimized set of 24 mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) loci, including a discriminatory subset of 15 loci, has recently been defined for the typing of Mycobacterium tuberculosis. Here, we evaluated the performances of this MIRU-VNTR typing system in combination with spoligotyping for the detection of transmission chains in a population-based study comprising 91% of culture-confirmed tuberculosis patients reported in 2003 in Hamburg, Germany. Of the 154 isolates investigated, more than 90% had high IS6110 copy numbers (>6). IS6110 restriction fragment length polymorphism (RFLP) typing resulted in 13 clusters, 5 of which had a confirmed epidemiological link. All five, as well as six of the eight IS6110 clusters with no identified epidemiological link, were perfectly matched by MIRU-VNTR typing with the 24 loci. Two IS6110 clusters were split by differences into 6 to 12 MIRU-VNTR loci, clearly supporting the absence of a link, as judged by contact tracing data. In contrast, only one MIRU-VNTR cluster, grouping what were probably epidemiologically unlinked isolates, was split by IS6110 RFLP. However, these isolates were also distinguished by spoligotyping. Both the optimized 24-locus and 15-locus sets thus showed a comparable to slightly better predictive value, especially when combined with spoligotyping, than the current gold standard IS6110 RFLP for the study of tuberculosis transmission in Hamburg. Because the epidemiological characteristics of this setting are similar to those of many developed countries, these results support the wide applicability of this real-time genotyping approach for population-based studies of M. tuberculosis transmission.Population-based molecular epidemiological studies of tuberculosis (TB) aim to identify pathways and predictors of ongoing TB transmission by determining the proportion of patients with identical isolates (clustering rate), as defined by molecular typing at regional or national scales. IS6110 restriction fragment length polymorphism (RFLP) typing of Mycobacterium tuberculosis is the gold standard method for this purpose (30). Consistently, M. tuberculosis outbreak isolates often show identical IS6110 RFLP patterns, and clustering by identical patterns is associated with well-identified risk factors for transmission, whereas unrelated patients often have isolates with different IS6110 fingerprints (see, for example, references 6, 21, 31, and 33).However, IS6110 fingerprinting is laborious and requires weeks of M. tuberculosis culturing, which limits the possibilities to use typing prospectively for more efficient TB control. Furthermore, comparison of the fingerprints from large data sets requires highly standardized experimental and computerized procedures (30). These problems complicate the determination of clustering rates in population-based studies and the exchange of data.Typing based on PCR amplification of multiple loci containing mycobacterial interspersed repetitive-unit-variable-number tandem repeats (MIRU-...