We conducted a population-based molecular epidemiological study of tuberculosis (TB) in Madrid, Spain (2002 to 2004), to define transmission patterns and factors associated with clustering. We particularly focused on examining how the increase in TB cases among immigrants in recent years (2.8% in 1997 to 1999 to 36.2% during the current study) was modifying transmission patterns. Mycobacterium tuberculosis isolates obtained from patients living in nine districts of Madrid (1,459,232 inhabitants) were genotyped. The TB case rate among foreign-born people was three to four times that of Spanish-born people, and the median time from arrival to the onset of treatment was 22.4 months. During the study period, 227 (36.3%) patients were grouped in 64 clusters, and 115 (50.7%) of them were in 21 clusters with mixed Spanish-born and foreign-born patients. Three of the 21 mixed clusters accounted for 21.1% of clustered patients. Twenty-two of 38 (57.9%) immigrants in mixed clusters were infected with TB strains that had already been identified in the native population in 1997 to 1999, including the three most prevalent strains. Factors identified as independent predictors of clustering were homelessness (odds ratio [OR], 2.3; 95% confidence interval [95% CI], 1.2 to 4.5; P = 0.011) and to be born in Spain (OR, 1.8; 95% CI, 1.2 to 2.6; P = 0.002). The results indicated that (i) TB transmission was higher in Spanish-born people, associated mainly with homelessness, (ii) that foreign-born people were much less likely to be clustered, suggesting a higher percentage of infection before arriving in Spain, and (iii) that an extensive transmission between Spanish- and foreign-born populations, caused mainly by autochthonous strains, was taking place in Madrid.