Background: Diarrhea causes 4% of all deaths and 5% of health loss lead to disability world-wide. Shiga-toxin-producing Escherichia coli can cause mild watery diarrhea to more serious complications from hemorrhagic colitis, and hemolytic uremic syndrome to even death. Present study was conducted to detect E. coli carrying Shiga toxin genes (stx1 and stx2) in E. coli isolated from patients with diarrhea in Hajar Hospital, Shahrekord, Iran.Methods: A total of 110 E. coli isolates collected from patients with diarrhea in 2011 were evaluated to investigate stx1, stx2 and eaeA genes by multiplex PCR.Results: The rate of stx1 and eaeA -positive E. coli isolates were 2.7% (3/110) and 1.8% (2/110), respectively. However, stx2 gene was not found in any isolate, eaeA was not also present in stx1 -positive E. coli isolates. Virulence Genes were not found in E. coli isolated from Women. The most antibiotic resistance rates of isolated virulence genes-positive E. coli were to Ampicillin and Trimethoprim and lowest antibiotic resistance was observed against Gentamicin and Chloramphenicol.Conclusion: The present study indicates that other infectious agents may play major role in the cause of diarrhea in the tested samples, especially in the women. Further studies on the causes of diarrhea are recommended.