Background: Molecular typing techniques are reliable tools for epidemiological study of tuberculosis because of their power in detecting recent transmission and differentiating reinfection and relapses. Objectives: The present study investigated epidemiological diversity among Mycobacterium tuberculosis strains circulating in three Khorasan provinces, Iran, using 12-loci MIRU-VNTR and Spoligotyping. Methods: This study was performed on 140 M. tuberculosis strains selected from the sputum of new cases of pulmonary tuberculosis patients in three Khorasan provinces, Iran. 12 loci MIRU-VNTR and Spoligotyping were performed on all isolates. Results: By MIRU-VNTR analysis, 76 distinct patterns comprising 19 clusters and 57 unique patterns were identified. Based on the results, MIRU10, MIRU26, and ETRF were highly discriminative, ETRD was poorly discriminative and other loci were designated as moderately discriminative. Spoligotyping of isolates revealed 51 distinct patterns: 26 patterns containing 33 strains (23. 6%) corresponding to orphan strains and 14 patterns containing 107 strains (76. 4%) corresponding to shared-types in the SITVIT2 database. Totally, 103 isolates (73. 6%) were classified into 14 clusters containing 2-56 isolates; the remaining 37 isolates (26. 4%) were unique patterns. By combining two techniques, 94 distinct patterns (15 clusters) contained 61 isolates (43. 6%), and 79 unique patterns were identified. The discriminatory power (HGDI) of combination of two techniques was 0. 962, which was higher than that of each technique alone. Based on the trees designed by Bionumerics software, we differentiated isolates with similar genetic patterns and grouped them together. Two great clusters were Haarlem and CAS lineage. All strains with combined drug resistance related to Beijing strains. Also, all mono drug-resistant strains related to Haarlem family; other strains were susceptible to the first-line antituberculosis drugs. Also, homoplasy was observed in a number of patterns. Conclusions: In MIRU-VNTR typing method, according to the genotype of each area, the loci with high discriminatory power (such as miru10, miru26, and ETRF in Iran) are recommended to be used and the loci with poor discrimination (such as ETRD in Iran) are not.