Tuberculosis (TB) is one of the main causes of death among curable infectious diseases and one of the top 10 causes of death worldwide. Hence, molecular typing of MTB strains is necessary for epidemiological studies and helps to identify risk factors for TB transmission. Therefore, the present study was conducted to determine molecular typing of drug-resistant M. tuberculosis strains isolated from Iran using the RFLP-PGRS method. Thirty-two MDR strains and one XDR strain were isolated from TB patients in four major cities of Iran. MTB isolates were subjected to drug susceptibility testing. Whole genomic DNA from mycobacterial colonies were extracted and hybridized with PGRS probe in RFLP analysis. All fingerprinted MDR and XDR isolates were grouped into 13 clusters. The largest cluster (cluster 3) contained 48.4% (n = 16) of all isolates. Clusters 1, 4, and 6 included 2, 4, and 2 isolates, respectively. Two isolates were in cluster 7, one was H37Rv standard strain, which was used as a control strain in this study, and eight isolates were placed in single clusters. This study provides information about molecular epidemiology of MDR-TB in Iran. The alarming increase in the incidence of MDR isolates, especially Beijing strains, raises concerns for TB control programs in Iran.