Background: Food poisoning (FP) caused by Clostridium botulinum is the most serious feature of FP in man consuming the contaminated food stuffs specifically seafood. The occurrence of the contamination to different species of the bacteria in four species of fish in Persian Gulf was investigated using selective culture and multiplex PCR (mPCR) assays. Methods: The samples were initially enriched in cooked meat broth for 7 days at 30 °C in an anaerobic condition followed by subculture onto blood agar. DNA of the samples were then extracted for further mPCR assay using three species-specific pair of primers to amplify the 782, 205 and 389 bp fragments corresponding to the A, B and E types of the microorganism (MO). Results: Out of 120 specimens, C. botulinum was detected in 5 samples (4.2%). Altogether, five and two samples were respectively positive in the mPCR and culture assays which were significantly different (p < 0.05). Conclusions: No evidence of type E was shown; which, was possibly resulted from the limitation of our study. Our results have suggested a reliable molecular assay to employ in the food Laboratories for rapid identification and differentiation of various kinds of C. botulinum.