The mycobacterial insertion sequence IS6110 has been exploited extensively as a clonal marker in molecular epidemiologic studies of tuberculosis. In addition, it has been hypothesized that this element is an important driving force behind genotypic variability that may have phenotypic consequences. We present here a novel, DNA microarray-based methodology, designated SiteMapping, that simultaneously maps the locations and orientations of multiple copies of IS6110 within the genome. To investigate the sensitivity, accuracy, and limitations of the technique, it was applied to eight Mycobacterium tuberculosis strains for which complete or partial IS6110 insertion site information had been determined previously. SiteMapping correctly located 64% (38 of 59) of the IS6110 copies predicted by restriction fragment length polymorphism analysis. The technique is highly specific; 97% of the predicted insertion sites were true insertions. Eight previously unknown insertions were identified and confirmed by PCR or sequencing. The performance could be improved by modifications in the experimental protocol and in the approach to data analysis. SiteMapping has general applicability and demonstrates an expansion in the applications of microarrays that complements conventional approaches in the study of genome architecture.