To analyze the strain distribution and clinical characteristics of nontuberculous mycobacteria (NTM) lung disease in Shanghai, a total of 90 samples from patients with NTM lung disease from Zhongshan Hospital, Fudan University from May 2017 to October 2018 were cultured by rapid automated mycobacterial culture and drug sensitive detection system (BACTEC MGIT960 System) or modified Roche culture method and identified by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The clinical data of patients were retrospectively collected. NTM strains were identified as 9 species, 65 cases of slow-growth NTM, most of which were Mycobacterium intracellulare (54.4%, 49/90); 25 cases of rapid-growth NTM, most of which were Mycobacterium abscessus (22.2%, 20/90). 67 cases were confirmed, 23 were suspected. The proportion of rare NTM in the confirmed patients was lower (6.0% vs 26.1%, P = 0.016). There was no significant difference in clinical manifestations between confirmed and suspected patients, but the proportion of confirmed patients with anti-NTM treatment history was significantly higher than that of suspected patients (85.1% vs 4.3%, P <0.001). Among the confirmed patients, the proportion of patients with rapid-growth NTM lung disease who had previous anti-tuberculosis treatment was significantly higher than that of the slow-growth group (52.9% vs 24.0%, P = 0.036). The results of this study provide a data reference for the clinical diagnosis and treatment of NTM lung disease.