We have developed an amplification-based reverse dot blot assay for the detection of specific sites of insertion of the Mycobacterium tuberculosis insertion sequence IS6110. In this assay, a set of biotin-labeled amplicons representing the various copies of IS6110 and their flanking sequences is generated by linker-mediated PCR. The amplicons are then hybridized to immobilized oligonucleotide probes that are specific for known IS6110 insertion sites. The method was evaluated using an array of oligonucleotide probes corresponding to IS6110 insertion sites from M. tuberculosis strains CDC1551, Erdman, and H37Rv, and multidrug-resistant strain W. A set of 72 DNA samples from 60 M. tuberculosis clinical isolates was analyzed for the presence or absence of these insertion sites, and the assay was found to be highly reproducible. This method of identifying insertion sites has been named “insite” and can be used for the genotyping of M. tuberculosis complex strains based on IS6110 insertion site profiles.