Background China is one of the high burden countries of Mycobacterium tuberculosis (TB) infection globally,with highincidence and mortality.We studied the molecular characteristics of rifampin (RIF) and isoniazid (INH) resistantMycobacterium tuberculosis strains from Beijing,China,in order to find out the genetic marker for rapid detection ofspecific drug resistance.Methods Forty pansusceptible and 81 resistant strains of Mycobacterium tuberculosis isolated from Beijing,Chinaduring 2002-2005 were analyzed.The modified rifampin oligonucleotide (RIFO) assay based on reverse line blothybridization was used to detect mutations in the 81 bp hot-spot region of rpoB gene,which is associated with RIFresistance.The INH resistance associated genes,regulatory region mab-inhA (-15C/T) and structural gene katG S315Twere detected by reverse line blot hybridization and PCR-restriction fragment length polymorphism (RFLP) methodrespectively.All the strains were typed by spoligotying and the Beijing genotype was further subdivided by NTF locusanalysis.The distribution of drug resistance associated mutations in the above genes was compared in these groups.Results Sixty-five (91.5%) of 71 RIF resistant and 52 (92.9%) of 56 multidrug-resistant (MDR,i.e.resistant to at leastRIF and INH) strains were found to harbor mutations in the rpoB hot-spot region.No mutation was detected in RIFsensitive strains.The specificity and sensitivity of the modified RIFO assay were 100% and 91.5%,respectively,katG315AGC>ACC and inhA-15C>T mutations were found in 40 (60.6%) and 10 (15.2%) of 66 INH resistant strains,respectively;7.6% of INH-resistant strains had mutations in both of these genes.Therefore,a combined use of both katG315 andinhA-15 identified 68.2% of INH-resistant strains.The Beijing genotype accounted for 91.7% of total strains and wasfurther subdivided into“modern”(76.6%) and“ancestral”(23.4%) group.There is no significant difference between“ancestral”and“modern”group in prevalance of drug resistance-associated gene mutations.Conclusions The hot-spot region of rpoB gene can be used as genetic marker for detection of RIF resistant strains;acombined use of both katG315 and inhA-15 can improve the detection rate of INH resistant strains;the Beijing genotypeis prevalent in Beijing,China;the modified RIFO assay can be a practical tool for rapid detection of RIF resistant andMDR isolates in the routine diagnostic work. Background China is one of the high burden countries of Mycobacterium tuberculosis (TB) infection globally, with highincidence and mortality. We studied the molecular characteristics of rifampin (RIF) and isoniazid (INH) resistant Mycobacterium tuberculosis strains from Beijing, China, in order to find out the genetic marker for rapid detection of specific drug resistance. Methods Forty pansusceptible and 81 resistant strains of Mycobacterium tuberculosis isolated from Beijing, Chinaduring 2002-2005 were analyzed. The modified rifampin oligonucleotide (RIFO) assay based on reverse line blothybridization was used to detect mutations in the 81 bp hot-spot region of rpoB gene, which is associated with RIF resistance. The INH resistance associated genes, regulatory region mab-inhA (-15C / T) and structural gene katG S315 Twere detected by reverse line blot hybridization and PCR- restriction fragment length polymorphism (RFLP) methodrespectively.All the type were by type spoligotying and the Beijing gen otype was further subdivided by NTF locusanalysis. The distribution of drug resistance associated mutations in the above genes was compared in these groups. Results Sixty-five (91.5%) of 71 RIF resistant and 52 (92.9%) of 56 multidrug-resistant , ieresistant to at least RIF and INH) were found to harbor mutations in the rpoB hot-spot region. No mutation was detected in RIFsensitive strains. The specificity and sensitivity of the modified RIFO assay were 100% and 91.5%, respectively, katG315AGC > ACC and inhA-15C> T mutations were found in 40 (60.6%) and 10 (15.2%) of 66 INH resistant strains, respectively; 7.6% of INH-resistant strains had mutations in both of these genes. use of both katG315 andinhA-15 identified 68.2% of INH-resistant strains. The Beijing genotype accounted for 91.7% of total strains and wasfurther subdivided into “modern ” (76.6%) and “ancestral ” (23.4%) group There is no significant difference between “ancestral ” and “modern ” group in prevalanceof drug resistance-associated gene mutations. Conclusions The hot-spot region of rpoB gene can be used as a genetic marker for detection of RIF resistant strains; acombined use of both katG315 and inhA-15 can improve the detection rate of INH resistant strains; the Beijing genotypes prevalent in Beijing, China; the modified RIFO assay can be a practical tool for rapid detection of RIF resistant and MDR isolates in the routine diagnostic work.