ABSTRACTThe increasing incidence of extensively drug-resistant (XDR)Mycobacterium tuberculosisin high-tuberculosis-burden countries further highlights the need for improved rapid diagnostic assays. An increasing incidence of XDRM. tuberculosisstrains in Pakistan has been reported, but drug resistance-associated mutations in these strains have not been evaluated previously. We sequenced the “hot-spot” regions ofrpoB,katG,inhA,ahpC,gyrA,gyrB, andrrsgenes in 50 XDRM. tuberculosisstrains. It was observed that 2% of rifampin, 6% of isoniazid, 24% of fluoroquinolone, and 32% of aminoglycoside/capreomycin resistance in XDRM. tuberculosisstrains would be undetected if only these common hot-spot regions were tested. The frequencies of resistance-conferring mutations were found to be comparable among all XDRM. tuberculosisstrain families present, including the Central Asian Strain, Beijing, and East African Indian genogroups and the Unique isolates. Additional genetic loci need to be tested for detection of mutations conferring fluoroquinolone, aminoglycoside, and capreomycin resistance in order to improve molecular diagnosis of regional XDRM. tuberculosisstrains.