BackgroundNon-tuberculous mycobacteria (NTM) represent a diverse group of environmental mycobacteria, with certain species implicated in infections affecting multiple tissues, including the lungs. The incidence of NTM-related diseases has been on the rise globally, particularly in developing countries. This study aimed to systematically investigate the ecological distribution, molecular identification, and antibiotic susceptibility profiles of NTM isolates in Hainan Island, furnishing a scientific basis for local control and prevention strategies.MethodsFrom 2015 to 2018, 158 suspected NTM samples were collected across 14 cities on Hainan Island. Following standardized sample processing and DNA extraction, molecular identification was conducted through PCR amplification of the rpoB and hsp65 genes, and sequencing, with species confirmation via GenBank database comparison. Phylogenetic analysis was performed to elucidate interspecies relationships and drug susceptibility testing was carried out for five antibiotics on M. abscessus isolates.Results118 NTM isolates were successfully identified, encompassing 24 distinct species. M. abscessus and M. intracellulare, accounting for 23.73% and 22.03% respectively, were the most prevalent. Phylogenetic analysis revealed clusters indicative of pathogenic and potentially pathogenic traits among different NTM species, with M. abscessus and M. fortuitum clustering as rapidly growing, pathogenic mycobacteria. Drug sensitivity testing indicated universal resistance of M. abscessus to doxycycline, yet high sensitivity to amikacin (71.4%) and linezolid (50.0%). Additionally, non-NTM species such as Nocardia thailandica, and Tsukamurella paurometabola were detected, suggesting potential misdiagnosis in clinical settings.ConclusionThis study provides a comprehensive dataset on the ecological distribution, molecular characterization, and antibiotic resistance of NTM in Hainan Island, highlighting the dominance of M. abscessus and M. intracellulare in local NTM infections.