Mutations in the katG locus of catalase peroxidase in Mycobacterium tuberculosis (MTB) account for major isoniazid (INH) resistance. In the South China region, a collection of 906 respiratory specimens and 142 MTB isolates was used to evaluate the sensitivity and specificity of a PCR-RFLP method for the detection of INH resistance-associated mutations. Except for four catalase-negative MTB isolates, katG PCR for a 620-bp amplicon was successful for all purified MTB isolates. For respiratory specimens, diagnostic sensitivity and specificity of katG PCR was 85 and 100 %. Subsequent RFLP of the katG amplicons by MspI digestion identified that 51 % of INH-resistant MTB were associated with the Thr315 phenotype, and that codon 463 was a polymorphic site with no linkage to INH resistance. The Arg463 wild-type MTB isolates predominant in the Western world were replaced by isolates carrying Leu463 in the South China region. RFLP patterns of katG amplicons from respiratory specimens were identical to those of the corresponding MTB cultured colonies. This method has potential application for rapid diagnosis of INH resistance due to katG Ser315Thr mutation.