We applied MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing) to directly analyze the bacilli present in 61 stain-positive specimens from tuberculosis patients. A complete MIRU type (24 loci) was obtained for all but one (no amplification in one locus) of the specimens (98.4%), and the allelic values fully correlated with those obtained from the corresponding cultures. Our study is the first to demonstrate that real-time genotyping of Mycobacterium tuberculosis can be achieved, fully transforming the way in which molecular epidemiology techniques can be integrated into control programs. ; Evaluation Studies; Journal Article; Research Support, Non-U.S. Gov't; María Alonso is supported by a contract (REF CA09/00054) from the Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias) and provides technical support in the Unidad Central de Análisis Molecular of Hospital General Universitario Gregorio Marañón. LightCycler 2.0 was acquired with a grant (REF IF08/36173) from Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias). The 3130xl Genetic Analyzer was partially financed by grants from Fondo de Investigaciones Sanitarias (IF01-3624, IF08-36173). The study was partially financed by Fondo de Investigaciones Sanitarias (S09/02205), Junta de Andalucía (PI-0444/2008 and PI-0306-2009), and SEPAR (763-09).