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Objective To investigate species distribution, antimicrobial susceptibility and possible transmission of non-tuberculous mycobacteria among Danish cystic fibrosis patients. Methods Thirty-five non-tuberculous mycobacteria cultured from 2006 to 2013 from Danish cystic fibrosis patients from two centres were investigated (one isolate per patient). The isolates were subjected to antimicrobial susceptibility testing to 15 antimicrobial agents by broth microdilution and to PCR for the erm(41) gene. Partial sequencing of the rpoB gene was used to determine species identification of the isolates. Results Twenty-three isolates were identified as Mycobacterium abscessus subspecies abscessus , 10 isolates as Mycobacterium abscessus subspecies bolletii (six isolates as subtype massiliense and four as subtype bolletii ), and two as Mycobacterium chelonae . Conclusion Strain relatedness was assessed by a Variable Number Tandem Repeat (VNTR) typing scheme which only revealed a few small clusters (2–4 patients) of related strains within the patient population. These clusters will be further investigated by whole-genome sequencing. [ABSTRACT FROM AUTHOR]