Background: The non-tuberculous mycobacteria (NTM) strains are important in the diagnosis of pulmonary diseases. However, the different source of the sample may correspond to different characteristics of both NTM disease and infected patients and these aspects have not been investigated.Methods: Samples isolated from different sources in inpatients with NTM strains were comprehensively analyzed in this study, including basic information, laboratory examination and coexisting diseases.Results: A total of 242 inpatients with NTM strains were considered in this study. Sputum (n=129) and bronchoalveolar lavage fluid (BALF, n=50) were the most common samples among patients with NTM strains from whom a sample was collected from a single source (n=179). The patients with NTM-positive BALF differed in age, gender, acid fast assay, the percentage of presence of chronic obstructive pulmonary disease (COPD) and bronchiectasis, and symptom duration compared to the patients with NTM-positive sputum. The patients with COPD were older and mainly male, with longer symptom duration, with higher rates of smoking history and prostatic hyperplasia than those without COPD. Patients with bronchiectasis were mainly females, with a lower percentage of them with a tuberculosis history and smoking history compared to patients without bronchiectasis. Excluding the patients with COPD and bronchiectasis, patients with NTM-positive sputum were older, with positive results in acid-fast assay and T. spot assay compared to patients with NTM-positive BALF.Conclusions: The different specimen source in NTM patients was associated with different patients’ characteristics, and its choice could be influenced by presence of COPD and bronchiectasis. Patients with COPD and bronchiectasis differed in gender and smoking history, thus, these aspects should be comprehensively analyzed during the investigation of patients with pulmonary NTM infection.