For molecular typing, the colonies from each culture positive slant were first subcultured into Löwenstein-Jensen culture media and then the single colonies were used for DNA extraction. By spoligotyping, 96% of strains were classified into four clusters. The largest genotypes were T superfamily with 56 isolates (68.29%), followed by Delhi/CAS (11, 13.41%), Beijing (six, 7.32%), and Haarlem (six, 7.32%) lineages. The other identified classes were Canetti (one, 1.2%), NEW-1 (one, 1.2%), and Uganda family (one, 1.2%). A combination of both MIRU-VNTR with spoligotyping increased the level of discrimination between isolates. For example, a T superfamily with 56 isolates by spoligotypes subdivided into 11 clusters (n = 40 strains) and 16 single isolates. In total, 65 strains (79.2%) classified into 18 clusters by using both PCR-typing methods. Minimum and maximum number of isolates per cluster ranged from two to eight strains. From an epidemiologic point of view, the majority of strains in clusters (13 of 18, 72%) were isolated from within one local area in the same county (Fig 2). Twenty-eight percent of those clustered were formed by isolates that were collected from different counties. Four of these clusters belong to the T superfamily with minimum (n = 2) and maximum (n = 5) differences in locus Mtub04, MIRU10, MIRU40, MIRU26, MIRU16, and ETR-A, respectively (Fig 3). As shown in Figures 1 and 3, the majority of interconnected clusters (n = 3 with 15 isolates) were collected from soil and water samples from two counties (Firuzkuh and Shahr-e-Ray). Overall, the allelic diversities of 15 MIRU-VNTR loci for soil and water isolates showed the locus MIRU 10 with highly discriminative power (> 0.6). Whereas loci Mtub04, MIRU40, MIRU16, ETR-A, MIRU26, MIRU31, QUB26, and QUB4156 were moderately discriminative (≥ 0.3, < 0.6), loci ETR-C, MIRU04, Mtub21, QUB11b, Mtub30, and Mtub39 were poorly discriminative loci (< 0.3) (Table 1).