BACKGROUND: Clostridium difficile (C. difficile) infection is one of the most important diseases in healthcare facilities and community. Ribotypes 027 and 078 are known as hyper-virulent strain of C. difficile in molecular study. PCR-ribotyping is a suitable method to interpret the relation of C. difficile isolated from food and hospital. Objectives: In the present study, the clostridim difficile binary toxin (cdtB) and ribotype pattern evaluated in toxigenic C. difficle isolated from beef. Methods: Detection of cdtB in 12 toxigenic C. difficile (encoding tcdA and tcdB gene) isolated from 100 beef samples was determined through PCR. Afterwards, PCR-ribotyping was performed to examine the ribotype patterns of C. difficile. Results: cdtB gene was not detected in any positive isolate. Ten different patterns were observed in 12 toxigenic isolates. No similarity existed in the ribotypes of our study with ribotypes 027 and 078. Conclusions: Albeit  ribotyp 027 and 078 were not found in our study, the isolation of toxigenic C. difficile with new ribotypes in Iran may indicate the probable hazard of this bacterium in public health. Comprehensive research about C. difficile in different food sources is recommended on a national level.