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Over the course of one year, a total of 120 human and 21 cattle isolates were tested using region of difference (RD)-based polymerase chain reaction\u00a0(PCR) and mycobacterial interspersed repetitive unites-variable number tandem repeats\u00a0(MIRU-VNTR). In <jats:italic>M. tuberculosis,<\/jats:italic> out of 120 isolates investigated, the most common genotype detected was NEW-1 (53.3%), followed by CAS\/ Delhi (24.1%), Haarlem (5%), Beijing (4.16%), Uganda I (4.16%), S (3.3%), Ural (0.83%), TUR (0.83%), Uganda II (0.83%), Lam (0.83%) and Cameroon (0.83%). The HGDI rate was 0.9981 and the clustering rate was 10.83. Of the isolates, QUB26 had the highest allele diversity (h: 0.76), while the loci Mtub29 and MIRU24 had the lowest (h: 0). In <jats:italic>M. Bovis<\/jats:italic>, out of\u00a0123 collected tissue samples, 21 (17%) grew on culture media. The HGDI rate was 0.71 and clustering rate was 85.7%. The locus ETRC had the highest allele diversity (h: 0.45). The findings of this study suggest that there is high genetic diversity among <jats:italic>M. tuberculosis<\/jats:italic> isolates in Khorasan Razavi Province, which is consistent with similar results from other studies in other provinces in Iran and neighboring countries. This indicates that the prevalent genotypes in this study are spreading in the Middle East region. Furthermore, considering that <jats:italic>M. 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