The objective of the present study was to investigate the diversity of mycobacterial isolates in a general hospital in Crete, Greece. 48 positive Lowenstein-Jensen cultures over a 3-y period were analysed by means of AccuProbe and GenoType assays. Non-tuberculous mycobacteria (NTM) comprised the majority of the isolates (56.3%, 27/48) vs 33.3% (16/48) of M. tuberculosis; 10.4% of the isolates could not be classified. Among NTM, M. lentiflavum was the predominant species isolated (9/27) followed by M. kansasii, M. gordonae and M. peregrinum, whereas no M. avium complex isolates were detected. This is the first detection of M. lentiflavum in Greece. The susceptibilities of the M. lentiflavum isolates to an extended panel of antibiotics were determined by the proportions method and the medical files of the 9 patients were reviewed. Three isolates were from urine, which is an unusual site. All strains exhibited multidrug resistance. The patients were adults with immunosuppression or predisposing conditions for NTM infection. Diagnosis of true infection was either not pursued or the patients died shortly after isolation.