Objective To investigate species distribution and antimicrobial resistance of non-tuberculous mycobacterial pulmonary disease (NTMPD), and provide basis for clinical diagnosis and treatment. Methods Positive clinical isolates cultured with BACTEC MGIT 960 system in a chest hospital in Hunan Province from 2017 to 2019 were collected, non-tuberculous mycobacteria (NTM) was isolated by MPB64 detection and PNB/TCH medium growth testing, NTM species were identified by 16Sr RNA, Hsp65 and ITS gene sequencing, resistance of Mycobacterium abscessus to 10 kinds of antimicrobial agents was detected with minimal inhibitory concentration (MIC) method. Results Among 10 443 positive strains reported by MGIT 960, 1 227 NTM strains were isolated, isolation rate was 11.7%. The top 4 strains were Mycobacterium intracellulare (27.8%), Mycobacterium abscessus (25.3%), Mycobacterium gordonae (16.5%) and Mycobacterium avium (16.1%). Susceptibility rates of Mycobacterium abscessus to amikacin, clarithromycin and linezolid were high, which were 94.6%, 87.5% and 82.1% respectively, while susceptibility rates to other antimicrobial agents were all lower than 50%. Conclusion Mycobacterium abscessus and Mycobacterium intracellular are the most prevalent strains of NTM in Hunan Province, amikacin and clarithromycin can be used as the preferred antimicriobial agents for clinical treatment of patients with Mycobacte-rium abscessus infection in Hunan Province.