Objective To understand the species distribution, antimicrobial resistance and clinical infection characteristics of non-tuberculous Mycobacteria (NTM) from suspected tuberculosis patients in Hainan area. Methods Sputum, alveolar lavage fluid and other respiratory tract specimens of suspected tuberculosis patients in a hospital of Hainan Province between January 2015 and August 2018 were collected, cultured and performed bacterial identification, NTM infection, antimicrobial susceptibility testing result and clinical data of patients with NTM pulmonary infection were analyzed. Results A total of 833 positive respiratory tract specimens were isolated from suspected tuberculosis patients, 126 of which were initially identified as NTM, identification of species showed that 3 strains were Mycobacterium tuberculosis, 3 were non-Mycobacterium, 4 were unable to be identified to species, among the other 116 pecimens, 118 strains of NTM were isolated. 109 cases were single NTM infection and 7 cases were mixed infection. There are 8 species of NTM, mainly Mycobacterium chelonae/Mycobacterium abscessus (41.5%) and Mycobacterium intracellular (37.3%). Eight NTM strains showed different degrees of resistance to 8 first-and second-line anti-tuberculosis drugs, the highest overall resistance rate was to isoniazid (96.6%), resistance rate to all 8 anti-tuberculosis drugs was as high as 32.2%. Incidence of NTM pulmonary infection in males was less than females, mainly in middle-aged and elderly people in Hainan coastal areas. The main clinical manifestations of patients with NTM pulmonary infection were cough and expectoration, chest tightness and shortness of breath, blood sputum or hemoptysis, loss of appetite and weight loss, underlying diseases included previous history of tuberculosis, bronchiectasis, pulmonary infection and hemoptysis, imaging manifestations showed secondary pulmonary tuberculosis, pulmonary cavity, pleural hypertrophy, bronchiectasis with emphysema and pulmonary infection. Conclusion NTM pulmonary infection in Hainan area is mainly caused by Mycobacterium chelonae/Mycobacterium abscessus and Mycobacterium intracellular, NTM is highly resistant to commonly used anti-tuberculosis drugs, NTM pulmonary infection and tuberculosis should be differentiated according to etiological examination.