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Background  India has the highest estimated burden of tuberculosis in the world, accounting for 21% of all tuberculosis cases world-wide. However, due to lack of systematic analysis using multiple markers the available information on the genomic diversity of  Mycobacterium tuberculosis  in India is limited.    Methodology/Principal Findings  Thus, 65  M. tuberculosis  isolates from New Delhi, India were analyzed by spoligotyping, MIRU-VNTR, large deletion PCR typing and single nucleotide polymorphism analysis (SNP). The Central Asian (CAS) 1 _DELHI sub-lineage was the most prevalent sub-lineage comprising 46.2% (n = 30) of all isolates, with shared-type (ST) 26 being the most dominant genotype comprising 24.6% (n = 16) of all isolates. Other sub-lineages observed were: East-African Indian (EAI)-5 (9.2%, n = 6), EAI6_BGD1 (6.2%, n = 4), EAI3_IND, CAS and T1 with 6.2% each (n = 4 each), Beijing (4.6%, n = 3), CAS2 (3.1%, n = 2), and X1 and X2 with 1 isolate each. Genotyping results from five isolates (7.7%) did not match any existing spoligopatterns, and one isolate, ST124, belonged to an undefined lineage. Twenty-six percent of the isolates belonged to the TbD1+ PGG1 genogroup. SNP analysis of the  pncA  gene revealed a CAS-lineage specific silent mutation, S65S, which was observed for all CAS-lineage isolates (except two ST26 isolates) and in 1 orphan. Mutations in the  pncA  gene, conferring resistance to pyrazinamide, were observed in 15.4% of all isolates. Collectively, mutations in the  rpoB  gene, the  katG  gene and in both  rpoB  and  katG  genes, conferring resistance to rifampicin and isoniazid, respectively, were more frequent in CAS1_DELHI isolates compared to non-CAS_DELHI isolates (OR: 3.1, CI95% [1.11, 8.70], P = 0.045). The increased frequency of drug-resistance could not be linked to the patients' history of previous anti-tuberculosis treatment (OR: 1.156, CI95% [0.40, 3.36],  P  = 0.79). Fifty-six percent of all new tuberculosis patients had mutations in either the  katG  gene or the  rpoB  gene, or in both  katG  and  rpoB  genes.    Conclusion  CAS1_DELHI isolates circulating in New Delhi, India have a high frequency of mutations in the  rpoB  and  katG  genes. A silent mutation (S65S) in the  pncA  gene can be used as a putative genetic marker for CAS-lineage isolates.

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In-Depth Molecular Characterization of Mycobacterium tuberculosis from New Delhi – Predominance of Drug Resistant Isolates of the ‘Modern’ (TbD1−) Type