Tuberculosis is a global pandemic and the incidence of nontuberculous mycobacteria (NTM) appears to be increasing worldwide. In this sense it is necessary that laboratories to use a rapid, accurate test, not laborious which does not require biosecurity measures. Molecular tests are accurate method, but is needs special equipments and lots labour, time and not economically. The aim for this study was to characterization the isolated of mycobacteria by immunochromatographic test, SD BIOLINE TB Ag MPT64 for differentiation into Mycobacterium tuberculosis (MTBC) complex and NTM. The immunochromatographic assay was evaluated with four reference strains of mycobacterial species and other clinical isolates of mycobacteria using the conventional methods as the gold standard. The results indicate that Ag MPT64 can be easily used for rapid discrimination between MTBC and NTM to con irm Mycobacterium tuberculosis speci ically obtained from early stages of subculture without any technical complexity.