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Background and Objectives: Shiga toxin-producing Escherichia coli (STEC) have emerged as human pathogens and contamination via animal origin has been a major public health concern. We compared the distribution of phylogenetic groups and prevalence of stx gene variants among the pathogenic strains of Escherichia coli isolated from feces of diarrheatic calves in Tehran suburb farms. Materials and Methods: In this study we screened 140 diarrheatic calves (1–15 days old) for E. coli strains during a 3 months period of time. The isolated strains were grouped into different phylotypes according to the presence of chuA, yjaA and TSPE4.C2 genes. Then, the prevalence of stx gene subtypes was evaluated in the B1 phylotypes. Results: From diarrheatic calves, 51 bacterial isolates were biochemically identified as E. coli and 31 isolates out of 51 were considered B1 phylotype using DNA Microarray technology. Of these isolates, 20 contained stx1a and stx1b and one harbored all mentioned variants of stx genes except stx2b2. Conclusion: This study showed that in Tehran suburb, the B1 phylotype of E. coli is prevalent as a causative agent of diarrhea in calves and the prevalence of stx1 gene subtypes is dominant in comparison with other subtypes. Considering the possibility that these stx genes can be spread to other strains, bovine E. coli strains are an important source of stx genes for other strains and further study and surveillance seems to be required for the exact identification of virulence profile of E. coli phylotypes in different hosts.