The aim of this study was to identify virulent Shiga toxin-producing Escherichia coli (STEC) strains isolated from faecal samples of 100 clinically healthy calves. In the present study, a total of 100 Escherichia coli (E. coli) isolates from clinically healthy calves belonging to 6 different farms located in Khorasan Razavi province, Iran, were examined for presence of virulence genes characteristic for STEC strains. Duplex PCR assay was used in order to determine the presence of the stx1, and stx2 genes. The stx1 or stx2 positive isolates was later analyzed with primers specific for eaeA, hlyA genes. PCR assay was also conducted on these isolates for detection of O157: H7 serotype. Our results showed that 15 isolates (15%) were positive for stx1 gene, 19 isolates (19%) were positive for stx2 gene and 8 isolates (8%) were positive for both stx1, and stx2. Totally 26 isolates were positive for at least one of stx1or stx2 genes. Among 26 isolates, which were tested for the presence of eaeA and hlyA genes, 5 and 22 of them were positive for these genes, respectively. Four isolates were positive for both eaeA and hlyA genes. Among 26 isolates which were analyzed for genes coding O157 and H7 antigens, one and four isolates were positive for O157 and H7 antigen gene, respectively. None of the isolates were positive for both antigen genes. Faecal contamination due to poor hygiene is a risk factor in contamination of meat and milk products.