A commercial line probe assay kit (Inno-LiPA Rif.TB) for rapid identification of mutations in the rpoB gene associated with rifampin resistance in Mycobacterium tuberculosis was evaluated with a collection of 51 rifampin-resistant strains. Nine distinct rpoB mutations were identified. Concordances with automated sequence results for five wild-type kit probes and four probes for specific mutations were 94.1 and 100%, respectively. Overall concordance of the line probe assay kit with phenotypic rifampin susceptibility testing results was 90.2%.