Background/aim: The purpose of the present study was to determine the distribution and epidemiological features of mycobacteria with molecular methods. Materials and methods: Fifty-five culture-positive samples were analyzed by polymerase chain reaction-restriction enzyme length polymorphism (PCR-RFLP) at species level, and their molecular typing was performed with spoligotyping. The IS6110 region and the locus of gene coding for Hsp65 were amplified. RFLP profiles were obtained by cutting the Hsp65 region with the Hae III and BstE II (Eco91I) enzymes. Spoligotyping was carried out by commercial kit. The H37Rv strain was used as the control. Results: All samples showed the same cutting pattern with the H37Rv strain. The RFLP profiles of 9 strains identified as "mycobacteria other than tuberculosis" were compatible with the M. tuberculosis complex. Spoligotyping of 55 isolates detected 13 different genetic profiles. The Beijing genotype was not detected. One isolate was described as an orphan strain according to the SpolDB4 database. The most frequently detected family was T1 with 32 strains (64%), followed by 9 isolates (18%) belonging to the LAM7 TUR family. Conclusion: PCR-RFLP is a specific, rapid, and effective method in routine diagnosis of mycobacteria. Spoligotyping is an ideal method in the determination of genotypic varieties of mycobacteria.