Tuberculosis (TB) is one of the most devastating infectious diseases that is highly endemic in Pakistan. Pakistan is ranked 5th amongst 22 high tuberculosis burden countries of the world. Global as well as national tuberculosis control program is further challenged by the spread of multiple drug resistant strains of Mycobacterium tuberculosis, the causative agent of tuberculosis. For controlling the spread of M. tuberculosis isolates, circulating in this region, it is important to explore the transmission dynamics and characteristics of these strains. This information, in turn, can help to implement better treatment and control measures. The study provides the information about the population structure of M. tuberculosis isolates in Pakistan. DNA fingerprinting of the strains was performed by high throughput spoligoriftyping and 24 MIRU-VNTR typing techniques. CAS family constituted the dominant group of the strains followed by the T family and EAI family while Beijing family showed the low prevalence. However, EAI strains were found to show high prevalence in Eastern part of the country. Molecular epidemiological methods play an important role to identify appropriate public health interventions and to measure their impact. Despite of the fact that Pakistan is harboring high disease burden, no molecular epidemiologic studies have yet been conducted to assess the disease transmission. Our study explores, for the first time, TB epidemiology in the Punjab province of Pakistan, using the gold standard tools of molecular epidemiology. We document a relatively low disease transmission rate in the population. The study also provides a good assessment of the discriminatory powers of the various genotyping techniques and suggests the use of duplex format of MIRU-VNTR typing to be used as genotyping technique in this setting because of its low cost and relatively less turnaround time as compared to simplex format. We further suggest the use of Qub 26, MIRU 10, Mtub 04, MIRU 26, MIRU 31 (ETR E), MIRU 16, Qub 4156 and Mtub 21 to be used as preliminary ‘fast lane’ screen to differentiate the M. tuberculosis strains in this particular geographical setting. The use of high throughput techniques like spoligoriftyping is recommended to be used as good tool to help the TB control programs in high disease burden countries. This powerful technique also helped to identify unreliable standards of phenotypic drug sencitivity testing (DST) in some local hospitals. Besides this, an in-house low cost test platform is configured and validated for the rapid screening of multiple drug resistance (MDR) in the present study. Being highly sensitive and specific, not only in the culture isolates but also in clinical samples, it could be used to screen MDR in point of care settings in the developing world where the need is acute. This study for the first time gives the comparative assessment of three freely available databases used to assign lineage to the M. tuberculosis isolates, uncovering the errors and inability of these databases in assigning lineages to isolates. Further, our study also pinpointed the defects in lineage assignation at sublineage level that arose due to the lack of database up gradation. The study also covered the assessment of occurrence of mutations at various target loci and their frequencies in M. tuberculosis isolates, resistant to first line anti- TB drugs. Overall, the profile of the mutations at various loci was similar to that found at other geographical locations worldwide. The most common mutations responsible for the rifampicin resistance were found in codon 531, 526 and 516 of rpoB gene, in isoniazid resistant isolates affecting the codon 315 of the katG and position -15 of the promoter region of inhA gene, in ethambutol resistant isolates affecting the codon 306 of embB gene and in streptomycin resistant isolates, targeting the codon 43 of rpsL gene and codon 512, 513 and 516 of rrs gene. In case of pyrazinamide, very few isolates showed mutations in targeted regions of pncA gene. Besides this, some novel mutations were also observed in this study. The relationship of specific mutations in rpoB and katG genes with M. tuberculosis lineages is also explored. The information about these mutations can be used to develop novel molecular diagnostic method that specifically could be implemented in Pakistan. However, prospective thorough epidemiological studies are needed to monitor continuously changing disease transmission dynamics in the community.