Abstract:[Abstract] The clinical Mycobacterium isolates were collected for the research to evaluate the effect for rapid species indentification of Mycobacterium clinical isolates. Primarily species were identified by culture method with differentiated PNB/TCH meida. By means of multi-locus polymerase chain reaction (Multi-locus PCR) the specific DNA fragments of 16SrRNA, Rv0577, IS1561, Rv1510, Rv1970, Rv3877/8 and Rv3120 genes in the genome of the isolates were examined respectively. The species were confirmed by rpoB-PRA and sequencing of hsp65 and rpoB. A total of 391 Mycobacterium isolates were collected for the species identification.By Multi-locus PCR, 378 were Mycobacterium tuberculosis, 6 were Mycobacterium africanum I type, and 7 were nontuberculous mycobacteria (NTM). 2 NTM confirmed By means of rpoB-PRA and sequencing of hsp65 and rpoB the 7 NTM strains were confirmed to one Mycobacterium avium,2 Mycobacterium marseille,and 4 Mycobacterium intracellular. The results of Multi-locus PCR were the same as that of rpoB-PRA and sequencing of hsp65 and rpoB. While by culture method with differentiated PNB/TCH media, 385 strains were Mycobacterium tuberculosis complex and 6 was NTM. Therefore, multi-locus PCR can identified Mycobacterium speices truly, reliably, simply and rapidly, and is significant useful value for the molecular epidemiology, clinical diagnosis and clinical therapy of the disease.