Objective: To understand the bacterial species distribution and relevant clinical characteristics of clinical isolates of 87 non-tuberculous Mycobacteria pulmonary disease (NTM-PD) patients in a specialized hospital of Nanjing. Methods: The strain distribution and related clinical characteristics of clinical isolates from patients with NTM-PD in the hospital from June 2017 to August 2018 were reviewed and analyzed retrospectively. Results: The NTM isolation rate was 15.5% in the specialized hospital in Nanjing, with a prevalence of 56.5%. NTM-PD patients were more frequent in males, especially in elderly male over 60 years old, while female patients were more common in 50 to 59 years old. The most common pathogenic strains were Mycobacterium intracellulare(70.1%), followed by Mycobacterium chelonei(11.5%) and Mycobacterium avium(11.5%). The common risk factors for NTM-PD patients were previous history of tuberculosis(64.4%), human immunodeficiency virus (HIV)positive(19.5%), bronchiectasis(19.5%), chronic obstructive pulmonary disease(COPD)(6.9%), hepatitis(5.7%), and Diabetes Mellitus(4.6%). The clinical symptoms of NTM-PD patients were non-specific respiratory symptoms, such as cough, sputum, fever, hemoptysis, chest distress and asthma, and there was no correlation between those clinical symptoms and bacteria species. However, asthma and hemoptysis were correlated with gender. The males were more prone to asthma, while females were more prone to hemoptysis. Hemoptysis and chest distress had relevance with the age. The older, the more likely hood of hemoptysis; while the younger, the more likely hood of chest distress. In the chest imaging manifestations, bronchiectasis was the most frequent, accounting for 39.1%, followed by pulmonary cavitation and nodules, accounting for 37.9%respectively, in which multiple thin-walled cavitation accounted for 90.9%. Conclusion: NTM-PD and tuberculosis have similar clinical manifestations. It's not easy to identify in clinical practice. It is necessary to alert the presence of NTM-PD with those who have previous history of tuberculosis, HIV-positive, bronchiectasis, chronic obstructive pulmonary disease, or chest imaging of bronchiectasis, pulmonary cavity and nodules. It is of great significance to identify the species as soon as possible for NTM patients in order to guide clinical treatment.