The increasing incidence of extensively drug-resistant (XDR) Mycobacterium tuberculosis in high-tuberculosis- burden countries further highlights the need for improved rapid diagnostic assays. An increasing incidence of XDR M. tuberculosis strains in Pakistan has been reported, but drug resistance-associated mutations in these strains have not been evaluated previously. We sequenced the "hot-spot" regions of rpoB, katG, inhA, ahpC, gyrA, gyrB, and rrs genes in 50 XDR M. tuberculosis strains. It was observed that 2% of rifampin, 6% of isoniazid, 24% of fluoroquinolone, and 32% of aminoglycoside/capreomycin resistance in XDR M. tuberculosis strains 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 XDR M. tuberculosis strain 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 XDR M. tuberculosis strains.