We performed spoligotyping of Mycobacterium tuberculosis isolates from 833 systematically sampled pulmonary tuberculosis (TB) patients in urban Mumbai, India (723 patients), and adjacent rural areas in western India (110 patients). The urban cohort consisted of two groups of patients, new cases (646 patients) and first-time treatment failures (77 patients), while only new cases were recruited in the rural areas. The isolates from urban new cases showed 71% clustering, with 168 Manu1, 62 CAS, 22 Beijing, and 30 EAI-5 isolates. The isolates from first-time treatment failures were 69% clustered, with 14 Manu1, 8 CAS, 8 Beijing, and 6 EAI-5 isolates. The proportion of Beijing strains was higher in this group than in urban new cases (odds ratio [OR], 3.29; 95% confidence limit [95% CL], 1.29 to 8.14; P ؍ 0.003). The isolates from rural new cases showed 69% clustering, with 38 Manu1, 7 CAS, and 1 EAI-5 isolate. Beijing was absent in the rural cohort. Manu1 was found to be more common in the rural cohort (OR, 0.67; 95% CL, 0.42 to 1.05; P ؍ 0.06). In total, 71% of isolates were clustered into 58 spoligotypes with 4 predominant strains, Manu1 (26%), CAS (9%), EAI-5 (4%), and Beijing (4%), along with 246 unique spoligotypes. In the isolates from urban new cases, we found Beijing to be associated with multidrug resistance (MDR) (OR, 3.40; 95% CL, 1.20 to 9.62; P ؍ 0.02). CAS was found to be associated with pansensitivity (OR, 1.83; 95% CL, 1.03 to 3.24; P ؍ 0.03) and cavities as seen on chest radiographs (OR, 2.72; 95% CL, 1.34 to 5.53; P ؍ 0.006). We recorded 239 new spoligotypes yet unreported in the global databases, suggesting that the local TB strains exhibit a high degree of diversity.