ABSTRACTWe have developed a multiplex assay, based on multiplex ligation-dependent probe amplification (MLPA), that allows simultaneous detection of multiple drug resistance mutations and genotype-specific mutations at any location in theMycobacterium tuberculosisgenome. The assay was validated on a reference panel of well-characterized strains, and the results show thatM. tuberculosiscan be accurately characterized by our assay. Eighteen discriminatory markers identifying drug resistance (rpoB,katG,inhA,embB), members of theM. tuberculosiscomplex (16S rRNA, IS6110, TbD1), the principal genotypic group (katG,gyrA), and Haarlem and Beijing strains (ogt,mutT2,mutT4) were targeted. A sequence specificity of 100% was reached for 16 of the 18 selected genetic targets. In addition, a panel of 47 clinicalM. tuberculosisisolates was tested by MLPA in order to determine the correlation between phenotypic drug resistance and MLPA and between spoligotyping and MLPA. Again, all mutations present in these isolates that were targeted by the 16 functional probes were identified. Resistance-associated mutations were detected by MLPA in 71% of the identified rifampin-resistant strains and in 80% of the phenotypically isoniazid-resistant strains. Furthermore, there was a perfect correlation between MLPA results and spoligotypes. When MLPA is used on confirmedM. tuberculosisclinical specimens, it can be a useful and informative instrument to aid in the detection of drug resistance, especially in laboratories where drug susceptibility testing is not common practice and where the rates of multidrug-resistant and extensively drug resistant tuberculosis are high. The flexibility and specificity of MLPA, along with the ability to simultaneously genotype and detect drug resistance mutations, make MLPA a promising tool for pathogen characterization.