ABSTRACTThe prevalence of nontuberculous mycobacterial diseases is increasing worldwide, and tuberculosis remains highly prevalent. Rapid and accurate microbial diagnoses of both tuberculosis and nontuberculous mycobacterial infections are required. A novel PCR–reverse sequence-specific oligonucleotide probe (PCR-rSSO) method-based mycobacterial detection panel (Myco-Panel) test was developed for the rapid identification of 30 mycobacterial species and subspecies. Clinical respiratory samples were collected from patients with suspected or confirmed tuberculosis and nontuberculous mycobacterial lung disease and those with other respiratory diseases. Myco-Panel tests were performed on the samples, and liquid mycobacterial culture and identification were performed for reference according to housekeeping gene sequences of mycobacteria in positive culture tubes. Furthermore, to assess the detection performance for several mycobacterial species rarely recovered in Japan, the accuracy of the Myco-Panel test was investigated using stock mycobacterial type strains and clinical isolates. A total of 178 clinical respiratory samples were analyzed. The Myco-Panel and sequence-based identification results for mycobacterial cultures were 83.1% concordant (kappa coefficient, 0.785 [95% confidence interval, 0.716 to 0.854]). The Myco-Panel correctly identified 281 of the 283 type strains and clinical isolates tested. The Myco-Panel test could accurately detect several mycobacterial species from clinical respiratory samples and mycobacterial suspensions. Rapid and accurate identification of pathogens using respiratory samples is possible using the Myco-Panel.IMPORTANCESpecies identification is important for the diagnosis of mycobacterial infections and decisions on treatment regimens. The Myco-Panel test accurately detects clinically common mycobacterial species that cause respiratory infections from clinical respiratory samples and mycobacterial suspensions. The rapid identification of multiple mycobacterial species will provide clinically useful information for the management of patients. Although we understand that the current diagnostic criteria require mycobacterial culture results in general for the diagnosis of nontuberculous mycobacterial infection, mycobacterial culture examination is a time-consuming process. The detection of potentially causative agents directly from clinical samples will aid in practical diagnosis and decision-making for rapid treatment initiation. This is a new laboratory method for species identification, and evaluating its performance is important.