The real-time PCR with duplex primer sets and the MPB64-based immunochromatographic assay are newly developed methods for rapid differentiation of mycobacteria. The aim of this study is to evaluate the two methods for differentiation between Mycobacterium tuberculosis complex and nontuberculous mycobacteria. A total of 95 clinical mycobacterial isolates belonging to 22 different species and 16 reference strains of 16 different species were differentiated by duplex real-time PCR method and MPB64-based immunochromatographic assay method. The two methods were evaluated by comparison with conventional biochemical technique as the gold standard method. The duplex real-time PCR method correctly differentiated all reference strains as well as the MPB64-based immunochromatographic assay method. For clinical isolates, the accuracy of the duplex real-time PCR method (100%) was slightly higher than the MPB64-based immunochromatographic assay method (97.9%), but there was no statistical significance between the two methods (P > 0.05), and there was an excellent agreement between them (Kappa = 0.957). The duplex real-time PCR method possesses greater potential for differentiation of mycobacteria in the clinical laboratory than the MPB64-based immunochromatographic assay method. However, the MPB64-based immunochromatographic assay method is more convenient than the duplex real-time PCR method when the number of sample is small.