A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid identification of rifampin (RFP)-resistant genotypes in Mycobacterium tuberculosis clinical isolates. The assay used the rpoB gene as target and was used to evaluate 148 clinical isolates (97 RFP-resistant isolates and 51 RFP-susceptible isolates). At the same time, the isolates were subjected to DNA sequencing and conventional drug susceptibility test. One hundred and forty one (95.3%) and 136 (91.9%) of the 148 strains were correctly identified by DNA sequencing and RDBH assay, respectively. None of the 51 RFP-susceptible isolates examined had alterations in rpoB. The sensitivity and specificity of the DNA sequencing were 92.8% and 100%, and the positive predictive value (PPV) and negative predictive value (NPV) were 100% and 87.9%, respectively. The sensitivity and specificity of the RDBH assay were 87.6% and 100%, and the PPV and NPV were 100% and 81.0%, respectively. Codons 531 and 526 of the rpoB were found to be the most common sites of nucleotide substitutions. Mutations at codons 511, 513, 515, 516, 517, 518, and 533 were also found. There were two-codon mutations in four isolates. No deletion and insertion was found in the rpoB gene. These results indicate that the RDBH assay is a rapid, simple, and reliable method for routine identification of RFP resistance in M. tuberculosis.