The present study aimed to find out the most prevalent lineage of Mycobacterium tuberculosis (MTB) and analysis of their mutation profile conferring to drug resistance at National Reference Laboratory (NRL) in New Delhi.Methods: A total 212 liquid culture MTB isolates of presumptive multidrug resistance tuberculosis (MDR-TB) patients were selected at NRL and subjected to whole genome sequencing (Illumina Miseq, USA). The genome sequencing data of 194 samples was successfully obtained in fastq files from instrument and analysed by Mykrobe predictor for lineage and mutation identification conferring drug resistance.Results: In a total 194 isolates, the number of isolates belonging to lineage and sub-lineage of lineage 1, 2, 3 and 4 were 13 (6.7%), 63 (32.4%), 82 (42.2%) and 28 (14.4%) respectively. Out of total 194 culture isolates analysed, 179 harbor at least one or more mutations. Among these 179 mutated isolates, following gene mutations were found in either single or in combinations: rpoB (146, 81.5%), katG (138, 77%), pncA (62, 34.6), gyrA (121, 67.5%), embB (106, 59.2%), rpsL (86,48%) followed by fabG1 (31, 17.3%), embA (18,10%), gid (6,3.3%), inhA (4), eis (4,2.2%), rrs (17, 9.4%). Lineage 2 was found to be most mutated MTB strains having atleast one or more mutations. In lineage 2, the frequency of rpoB, katG, gyrA, embB, and pncA gene mutations was 92%, 82.5%, 93.6%, 68.2%, and 61.9%, respectively. This was higher than in lineage 1 (30.7%, 53.8%, 15.2%, 38.4%, 0% respectively), lineage 3 (69.5%, 68.2%, 46.3%, 46.3%, 24.3% respectively), and Euro-American lineage 4 (74%, 62.9%, 48.1%, 40.7%, 26.9% respectively).Conclusions: Based on this study, lineage 3 may the most prevalent strain of MTB in north India. More attention required to take care patients having Lineage 2 infection to break chain of transmission as it was found to be most mutated/resistant strains.