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The clinical relevance of pulmonary non-tuberculous mycobacteria (PNTM) in The Gambia is unknown. The aim of this study was to estimate the prevalence of non-tuberculous mycobacteria (NTM) in colonisation, and the burden of clinically relevant pulmonary NTM (PNTM) disease in The Gambia. This was a cross-sectional study of the prevalence of NTM in participants aged ≥ 15 years, in a nationwide tuberculosis (TB) prevalence survey between December 2011 and January 2013. We enrolled 903 participants with suspected NTM and NTM cultures were confirmed by 16S rRNA gene sequencing analyses. We applied the American Thoracic Society/Infectious Disease Society of America (ATS/IDSA) diagnostic criteria to determine clinical relevance of NTM. A total of 575 participants had acid-fast bacilli (AFB) positive Mycobacterial Growth Indicator Tube (MGIT) cultures and 229 (39.8%) were NTM. M. avium complex was by far the most isolated NTM (71.0%), followed by M. fortuitum (9.5%) and M. nonchromogenicum (2.9%). Older participants (> 24 years old) were four times more likely to have NTM in their sputa. Only 20.5% (9/44) NTM cases met the ATS/IDSA criteria for NTM disease. This study provides important data on the prevalence of NTM in pulmonary samples of suspected TB cases with AFB positive cultures from a nationally representative population in The Gambia. Enhanced PNTM surveillance is recommended to better understand the contribution of NTM to pulmonary disease., (© 2022. The Author(s).)