The most prevalent strain of Mycobacterium tuberculosis in Madrid, Spain (strain 5) was recovered from 45 cases between 1997 and 2004 and showed a highly homogeneous genetic composition. This strain was not exclusive to Spain, and its spoligotyping signature (ST20) was found in entries from different countries in the SITVIT1 database. Patients infected with strain 5 were more frequently positive for human immunodeficiency virus and autochthonous, and had been in prison more frequently, but strain 5 did not show increased infectivity in an in-vitro model of infection.