For lowering prevalence of drug resistance it is necessary to diagnose TB in tuberculosis sputum suspect patients instead of TB-cultured samples which required a long time of culturing. Comparison of the results of drug resistant bacterial genes in both tuberculosis suspect sputum and multi-drug resistant DNA isolates detected by MAS-PCR. In the current study, the genetic mutations linked with INH, RIF as well as EMB drugs were detected by MAS-PCR simultaneously in MDR as well as TB suspect sputum isolates. 175/291 samples belonged to MDR and 116/291 samples belonged to tuberculosis suspect group. In all the isolates, presence of Mycobacterium tuberculosis-species (100%) was confirmed by targeting hupB gene. In MDR group, maximum prevalence of gene mutation was detected in rpoB531 (92.57%) and embB306 (97.71%) while in TB-suspect group, equal percentage (96.55%) of mutation was detected in rpoB531 and embB306 by MAS-PCR. Collectively, rpoB531 (n=274, 94.15%) and embB306 (n=283, 97.25%) mutation were observed in maximum tuberculosis cases. MAS-PCR technique yielded reliable results and showed massive Isoniazid, Rifampicin and Ethambutol drugs resistance in TB-isolates from Pakistan; hence it can be used in clinical laboratories with high burden of tuberculosis to detect drug resistance rapidly and cost effectively.