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Background Non-tuberculous mycobacteria (NTM) infection is an increasing problem worldwide. The epidemiology of NTM in most Asian countries is unknown. This study investigated the epidemiology, and clinical profile of inpatients in whom NTM was isolated from various anatomical sites in a Singaporean population attending a major tertiary referral centre. Methods Demographic profile, clinical data, and characteristics of patients hospitalized with NTM isolates at a major tertiary hospital over two-year period were prospectively assessed (2011–2012). Data collected included patient demographics, ethnicity, smoking status, co-morbidities, NTM species, intensive care unit (ICU) treatment, and mortality. Results A total of 485 patients (62.1% male) with 560 hospital admissions were analysed. The median patient age was 70 years. Thirteen different NTM species were isolated from this cohort. Mycobacterium abscessus (M. abscessus) (38.4%) was most frequently isolated followed by Mycobacterium fortuitum (M. fortuitum) (16.6%), Mycobacterium avium complex (MAC) (16.3%), Mycobacterium kansasii (M. kansasii) (15.4%), and Mycobacterium gordonae (M. gordonae) (6.8%). Most (91%) NTM was isolated from the respiratory tract. The three most common non-pulmonary sites were; blood (2.7%), skin wounds and abscesses (2.1%), and gastric aspirates (1.1%). A third (34.4%) of the study population had prior pulmonary tuberculosis (PTB). There was a significant association between isolated NTM species, and patient age (p=0.0002). Eleven (2.2%) patients received intensive care unit (ICU) treatment during the study period and all cause mortality within 1 year of the study was 16.9% (n=82). Of these, 72 (87.8%) patients died of pulmonary causes. Conclusions The profile of NTM species in Singapore is unique. M. abscessus is the commonest NTM isolated, with a higher prevalence in males, and in the elderly. High NTM prevalence is associated with high rates of prior PTB in our cohort. Published version