This article reports the identification of 57 AFB cultures isolated from clinical specimens by using traditional methods (TM, including biochemical and cultural methods) and modern ELISA with monoclonal antibody (McAb-ELISA) and nested primer gene amplification assay (NPGAA). The representive AFB culture M. A1, A7, A19, A21 and A22) isolated from human lepromas were identified as new species by TM and it was shown that they were not identical to M. leprae by McAb-ELISA and NPGAA. Among another set of samples (M. S17, S1, S2, S2R, S7, S29), M. S17 was identical to M. scrofulaceum as assessed by TM only, while the others were found to be similar to M. tuberculosis and different from M. leprae using TM and McAb-ELISA, and identical to M. tuberculosis with NPGAA. The authors conclude that TM and MM are very useful for identifying mycobacteria, while MM was much more sensitive and specific than TM. The selection and use of these methods depends on practical need.