We evaluated the clinical usefulness of spoligotyping, a polymerase chain reaction-based method for simultaneous detection and typing of Mycobacterium tuberculosis strains, with acid-fast bacilli-positive slides from clinical specimens or mycobacterial cultures. Overall sensitivity and specificity were 97% and 95% for the detection of M. tuberculosis and 98% and 96% when used with clinical specimens. Laboratory turnaround time of spoligotyping was less than that for culture identification by a median of 20 days. In comparison with IS6110-based restriction fragment length polymorphism typing, spoligotyping overestimated the number of isolates with identical DNA fingerprints by approximately 50%, but showed a 100% negative predictive value. Spoligotyping resulted in the modification of ongoing antimycobacterial treatment in 40 cases and appropriate therapy in the absence of cultures in 11 cases. The rapidity of this method in detection and typing could make it useful in the management of tuberculosis in a clinical setting.