Objective To establish multi-PCR-single strand conformational polymorphism analysis (mPCR-SSCP) for rapid detection of isoniazid (INH) and rifampin (RIF) resistance associated katG,inhA and rpoB genes of Mycobacterium tuberculosis isolates.Methods The INH-and RFP-resistance of 134 isolates was determined by using drug susceptibility test.The primers were designed for detecting INH and RFP resistance-associated katG,inhA and rpoB gene in the isolates by mPCR-SSCP.PCR-DS technique was applied to detect the mutations in katG,inhA and rpoB genes.All the results from different assays were subsequently analyzed as well as compared.Results All of the 134 tested isolates had katG,inhA and rpoB genes.Of the 134 isolates,42 (31.3％) and 45 (33.6％) strains were INH-and RFP-resistant,respectively.The results of mPCR-SSCP and PCR-DS showed that all the 92 INH-susceptible isolates had no mutation in katG and inhA genes with 100％ specificity.In the 89 RFP-susceptible isolates,2 and 1 had mutation in rpoB genes confirmed by mPCR-SSCP and PCR-DS with 97.8％ or 98.9％ specificity,respectively.Among the 42 INH-resistance isolates,33 and 36 strains had the mutations in katG and/or inhA genes due to the results of mPCR-SSCP and PCR-DS with 78.6％ or 85.7％ sensitivity,respectively.The results of mPCR-SSCP and PCR-DS also demonstrated that in the 45 RFP-resistance isolates,41 and 43 strains had the mutations in rpoB gene with 91.1％ or 95.6％ sensitivity,respectively.Conclusion The mPCR-SSCP established in this study can be used to rapidly detect INH and RFP-resistance associated mutations in katG,inhA and rpoB genes of M.tuberculosis with convenience,specificity and sensitivity,which shows a good prospect for application in clinic.