Effective tuberculosis (TB) control is hindered by lack of rapid diagnostic tests for detection of drug-resistant TB (DR-TB). Use of molecular tools for rapid detection of multi-and extensively- DR-TB, could facilitate early initiation of appropriate anti-tubercular treatment (ATT) regimen thereby interrupting transmission. Understanding the urgent situation, we standardized and evaluated 4 individual multiplex allele specific PCR (MAS-PCR) assays on 450 sputum specimens for Mycobacterium tuberculosis (MTB) detection and determination of drug resistance by targeting katG315, rpoB531, gyrA 94, rrs 1401 codon mutations for determination of resistance to Isoniazid (INH), Rifampicin (RIF), Fluoroquinolones (FQ) and Aminoglycosides (AG) respectively. Using a single sputum specimen, MAS-PCR correctly identified 97.2% (281/289; 95% CI:95–99) culture positive patients as MTB positive, 100% (271/271; 95% CI:99–100) for smear positive cases and 55.5% (10/18; 95% CI:34–75) for smear negative cases; and correctly identified 93.6% (104/111; 95% CI:87–97) of culture negative patients. Individual MAS-PCR assays reported variable diagnostic accuracy for determination of drug resistance. On comparison with phenotypic drug susceptibility testing, MAS-PCR assays correctly identified 89.2% (191/214; 95% CI:84–93), 94.9% (187/197; 95% CI:91–97), 72.5% (98/135; 95% CI:65–79) and 92.3% (24/26; 95% CI:75–99) of INH resistant, RIF resistant, FQ resistant and AG resistant specimens respectively; and correctly identified 94% (63/67; 95% CI:85–98), 86.9% (73/84; 95% CI:78–93), 93.1% (136/146; 95% CI:88–96) and 99.2% (253/255; 95% CI:97–100) of INH sensitive, RIF sensitive, FQ sensitive and AG sensitive specimens respectively. Thus, use of MAS-PCR assays for rapid detection of DR-TB is recommended, enabling early initiation of appropriate ATT.