To understand pathogen spectrum of nontuberculosis Mycobacteria (NTM) and the dominant NTM in Gansu Province and provide the scientific basis for the effective prevention and treatment of NTM diseases,875 Mycobacteria isolates were collected from 2012 to 2014 in Lanzhou Pulmonary Hospital,NTM species were identified by means of PNB/TCH differentiate medium and 16S rRNA gene sequence analysis respectively.Forty-six isolats of NTM were identied from 875 PNB/TCH.Then with 16S rRNA gene sequence analysis,the NTM strains were identified to 3 strains of Nocadia and 43 strains of NTM,including M.intracellulare,M.kansasii,M.avium,M.senegalense,M.gordonae,M.szulgai,M.peregrinumand M.fortuitum.Among them,there were 31 strains of M.intracellulare,which accounted for 72.09％ of the total number of NTM strains.The dominant nontuberculosis Mycobacteria in Gansu Province were mainly M.intracellulare.The application of molecular biology can rapidly and accurately identify the species of nontuberculosis Mycobacteria,and can provide relevant evidence for clinical diagnosis and therapy.