To evaluate the Multiplex Allele Specific-Polymerase Chain Reaction (MAS-PCR) assay for the detection of katGS315T mutations in Mycobacterium tuberculosis (MTB) clinical isolates. Totally, 78 MTB clinical isolates were analyzed by conventional Lowenstein–Jensen (LJ), BACTEC MGIT-960^TM liquid cultures, MAS-PCR, PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) and DNA sequencing methods. Fifty one (65.4%) out of 78 isolates were drug resistant, and 27 isolates (34.6%) were drug susceptible. MAS-PCR assay identified katG315 mutations in 47 (60.2%) isolates, in which 41 (52.5%) isolates had mutation at codon S315T (AGC®ACC), 6 (7.7%) isolates had mutation at codon S315N (AGC®AAC), and none of the 27 susceptible isolates had mutation. Concordant result was obtained by PCR-RFLP and DNA sequencing method was used further to validate the results obtained by MAS-PCR and PCR-RFLP assays. Novel mutations were found at G305V (GGC®GTC) and Q295P (CAG®CCG) codons in combination with S315T codon in 5 (6.4%) and 4 (5.1%) drug resistant strains respectively. katGS315T was the frequently mutated codon of katG gene in M. tuberculosis isolates, which can be used as a marker of choice for direct detection of isoniazid resistance by MAS-PCR assay in regions where katGS315T codon mutation is predominant among drug resistant isolates.