The aim of this study was to determine the prevalence of Clostridium botulinum in honey samples using conventional methods and multiplex PCR (mPCR). A total number of 150 honey samples were randomly collected from apiaries, retail shops, weekly open bazaars, and supermarkets in Samsun, Turkey. Of 150 honey samples, 4 (2.6%) were positive for the botulinum neurotoxin gene by mPCR analysis. A total of 4 C. botulinum isolates were obtained from the mPCR positive samples, of which 3 were type A and 1 was type B. No samples were positive regarding the type E and type F neurotoxin genes. This is the first report of type A and type B spores of C. botulinum being detected and isolated in Turkey. This study revealed that some honey samples may present a potential hazard for food borne and infant botulism.