Mycobacterium fortuitum is a clinically important species among nontuberculous mycobacteria (NTM). Treatment of diseases caused by NTM is challenging. The aim of this study was identification of drug susceptibility and detection of mutations in erm(39) related to clarithromycin resistance and in rrl related to linezolid resistance in clinical isolates of M. fortuitum in Iran. In the study, 328 clinical NTM isolates were subjected to identification based on rpoB and 15% of isolates were assigned to M. fortuitum. Minimum inhibitory concentration for clarithromycin and linezolid was determined by E-test. Altogether 64% of M. fortuitum isolates showed resistanc to clarithromycin and 18% of M. fortuitum isolates showed resistance to linezolid. PCR and DNA sequencing were performed in erm(39) and in rrl genes for detection of mutations related to clarithromycin and linezolid resistance, respectively. Sequencing analysis revealed (84.37%) single nucleotide polymorphisms in the erm(39). A total 55.55% of M. fortuitum isolates harbored an A-G, 14.81% harbored an C-A, 29.62% harbored an G-T mutation in erm(39) at position 124, 135, 275. Seven strains harbored point mutation in the rrl gene either at T2131C or at A2358G. Our findings showed M. fortuitum isolates have become a serious problem with high-level antibiotic resistance. The existence of drug resistance to clarithromycin and linezolid indicates more attention to the study of drug resistance in M. fortuitum.  Kulcsszavak: non-tuberculous mycobacteria, Mycobacterium fortuitum, clarithromycin, linezolid, drug resistance