A new PCR-reverse dot blot hybridization (RDBH) assay was developed for the rapid identification of Mycobacterium species in clinical isolates. The assay, which targets the 16S rRNA, was evaluated for 27 mycobacterial reference strains and 340 clinical isolates that were simultaneously identified by DNA sequencing and conventional methods, including growth characteristics, pigment production, colony morphology, and biochemical tests. All reference strains and clinical isolates hybridized to the Mycobacterium genus probe (probe M) on the membrane (100% sensitivity). Each probe had only one hybridization signal with the corresponding Mycobacterium species or complex (100% specificity). Compared with DNA sequencing, the RDBH assay correctly identified 337 (99.1% accuracy) of the 340 isolates tested. One M. asia isolate and one M. neoaurum isolate were not identified by the RDBH assay due to the absence of specific probes for the two species on the membrane. Three isolates with different nucleotide sequences from M. intracellulare reference strains had a negative hybridization signal with probe c, which is specific for M. intracellulare. The whole procedure can be completed within 2.5 h post-PCR processing. A total of 32 of 340 isolates were erroneously identified by conventional methods (90.6% accuracy). Molecular identification based on the 16S rRNA sequence was superior to the conventional approaches in speed, sensitivity, and specificity. Therefore, the RDBH assay can be considered a rapid, simple, and reliable method for routine identification of frequently occurring and clinically relevant mycobacteria.