AimsThis study aimed to develop a rapid, simple and cost-effective method for the differentiation of Mycobacterium species. Methods and ResultsA total of 80 clinical mycobacterial isolates belonging to 12 different species and 16 reference strains of 16 different species were differentiated by the simplex real-time PCR coupled with melting temperature calling analysis. By comparing their melting profiles with those of the reference strains, all clinical mycobacterial isolates were differentiated as Mycobacterium tuberculosis complex or nontuberculous mycobacteria, and the latter were further divided into five groups. In comparison with 16S-23S internal transcribed spacer sequencing method as the gold standard method, both sensitivity and specificity of the assay were 100% when it was used for the differentiation between Myco.tuberculosis complex and nontuberculous mycobacteria. ConclusionsThe simplex real-time PCR coupled with melting temperature calling analysis could be an alternative method for the differentiation between Myco.tuberculosis complex and nontuberculous mycobacteria. Significance and Impact of the StudyRapid differentiation of mycobacteria could shorten the diagnostic time of mycobacterial diseases. It is also helpful for achieving optimal therapy and appropriate patient management.