The stability of IS6110 restriction fragment length polymorphism (RFLP) pattern was determined in 31 isolates from patients with multidrug-resistant tuberculosis (MDR-TB). These patients were in actual chains of transmission and they referred to the National Institute of Tuberculosis and Lung Diseases, Tehran, Iran. Susceptibility testing against first- and second-line drugs were performed by the proportional method on Lowenstein-Jensen culture media. Thereafter, DNA fingerprinting by IS6110 with direct repeat (DR) region as a probe was performed by standard protocols. The rate of IS6110 changes was 16%, although, no variation was found in the DR region, in a time-span of 1-63 months. The strains with unstable IS6110 patterns were resistant to all drugs tested, and the majority of them (60%) were collected from HIV-positive patients. The results demonstrated that for a reliable interpretation of strain typing, it is better to use an additional marker along with IS6110 RFLP.