We developed a method to identify mycobacteria by sequencing hypervariable regions of the polymerase chain reaction-amplified 16S ribosomal RNA gene. This method is nearly specific for mycobacteria and uses positive culture from liquid or solid medium without the needfor lengthy subculture. It shortens identification time to 3 days, which is much faster than the conventional biochemical method (mean, 8 weeks). It applies to all mycobacteria (approximately 100 species), unlike current nucleic acid hybridization methods, which probe only 4 species. The identifications are the same or are species specific for the well-characterized mycobacteria (59/68 [87%]) or more accurate for recently proposed species (9/68 [13%]). The method requires a single sequencing reaction, which is efficient and cost-effective. Therefore, this method is clinically and academically useful.