To evaluate and compare the usefulness of IS6110-restriction fragment length polymorphism (RFLP) and spoligotyping in the epidemiology of tuberculosis in Iran, Mycobacterium tuberculosis complex strains, isolated in 2 different areas of Iran, were subjected to RFLP and spoligotyping. The average number of IS6110 copies per strain was 11 and ranged from 5 to 18 among the M. tuberculosis strains. In total, among the 62 isolates, 56 different patterns were observed. 50 strains had unique RFLP patterns (89%) and 12 (11%) revealed patterns that were found among at least 1 other isolate. Spoligotyping of 97 isolates resulted in 42 different patterns, of which 72% were found in 15 clusters. 14 (29%) out of 48 investigated isolates were resistant to 1 or more antituberculosis drugs and 57% of the resistant isolates were isolated from Afghan immigrants. Ten percent of the isolates represented the Beijing genotype, including 4 of the 14 (36%) resistant strains. Three of these resistant Beijing strains were isolated from Afghan patients. IS6110-RFLP typing could be useful for studying the epidemiology of tuberculosis in Iran. IS6110 patterns were polymorphic and the average IS6110 copy number was high.