Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens primarily associated with the consumption of contaminated ground beef and are an important food safety concern worldwide. STEC has been found to produce a family of related cytotoxins known as Shiga toxins (Stxs). Shiga toxins have been classified into two major classes, Stx1 and Stx2. A single strains of STEC can produce Stx1, Stx2 (or its variants) or both. The aims of this project were to determine the prevalence and molecular characteristics of STEC isolates from chicken flocks in Northwest of Iran. A total of 350 fecal samples from 28 broiler farms were screened for the presence of STEC by conventional culture methods and polymerase chain reaction (PCR). All samples were initially subjected to phenotypical analysis using the Sorbitol MacConkey agar plate for the detection of the sorbitol negative E. coli, and then for genotypic analysis, by multiplex PCR for detection of stx1and stx2 genes. STEC were isolated from 14 (4 %) chicken fecal samples. To our knowledge, this is the first report of isolation of STEC from poultry in Iran. To conclude, this work revealed the presence STEC strains harboring stx1 and stx2 gene in healthy chicken fecal samples in Northwest of Iran suggesting they can play as an important potential source of contamination for people working on broiler farms or are in contact with chicken carcasses at meat processing plants.