In the current study we aimed to execute a rather less complicated molecular tying method, i.e. the random amplification of polymorphic DNA (RAPD) analysis to find the heterogeneity of Iranian strains of Mycobacterium tuberculosis. The isolates comprised a total of 96 strains of M. tuberculosis collected from clinical specimens of patients in Isfahan and Tehran. The isolates were assigned to the species M. tuberculosis by the key conventional and molecular methods. They were then subjected to RAPD analysis by four arbitrary primers, namely, the primers 27F, 1525R, MS- GF and INS-2. They were then evaluated for the number and intensity of the band patterns. The RAPD profiles of the Iranian isolates showed a degree of heterogeneity which varied based on the primer used. However, analysis of the isolates by primer INS-2 revealed the highest degree of diversity yielding 31 distinguishable RAPD types. RAPD analysis provides a rapid and easy means of identifying heterogeneity among the M. tuberculosis isolates. This typing system might be considered a valuable alternative molecular typing for countries with limited resources provided that the reproducibility and reliability of the method is carefully assured.