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Molecular detection of rifampin resistance (rpoB analysis) in Mycobacterium leprae was determined for 49 patients who experienced relapse of multibacillary leprosy and for 34 untreated patients. Molecular detection of ofloxacin resistance (gyrA analysis) was determined for the 12 patients who experienced relapse and who had received ofloxacin. Results of molecular tests were compared with the reference susceptibility test in the mouse footpad. Overall, the efficiency of molecular detection—that is, positive DNA amplification—was 95%, whereas that of the in vivo test was 55% (P > .001). Results of molecular detection and in vivo test were fully concordant when both were available—that is, for 35 rifampin-sensitive cases of leprosy (no rpoB mutation), 4 ofloxacin-sensitive cases (no gyrA mutation), 11 rifampin-resistant cases (rpoB missense mutations), and 1 ofloxacin-resistant case (gyrA mutation). rpoB and gyrA analysis appears to be an effective method for detection of rifampin and ofloxacin resistance in patients with leprosy.