A simple, rapid, and nonradioactive method for routine detection of fluoroquinolone resistance in mycobacteria is described. A single-strand conformation polymorphism (SSCP) methodology, based on the use of mini-gels and silver staining of DNA, was optimized for the analysis of denatured DNA products obtained by polymerase chain reaction (PCR) from the gyrA gene involved in fluoroquinolone resistance in mycobacteria. The method was successfully applied to fluoroquinolone-susceptible and -resistant laboratory strains of Mycobacterium smegmatis and to clinical strains of Mycobacterium tuberculosis isolated from patients who developed resistance during the course of fluoroquinolone treatment.