Mutations in the rpoB gene of 90 rifampin-resistant Mycobacterium tuberculosis isolates mostly from Asian countries were analyzed. Ten distinct single-nucleotide substitutions were found among the isolates by automated sequencing. A 3-nucleotide insertion was found in two isolates, and no mutation was found in five isolates (5.6%). A reverse hybridization-based line probe assay (INNO-LiPA Rif TB) for rapid detection of the mutations was evaluated with these isolates. Concordance rates with sequencing results for five wild-type probes (S probes) and four probes for specific mutations (R probes) were 96.7 and 100%, respectively. The overall concordance rate with the in vitro susceptibility testing results was 92.2% (83 of 90 isolates). These results indicate that a commercial line probe assay kit may be useful for rapid diagnosis of rifampin-resistant tuberculosis.