The aim of this study was examination of 240 multifloral honey samples collected from Polish apiaries for C. botulinum occurrence. Honey was collected directly from apiaries after the extraction process. Samples were inoculated by using the dilution and centrifugation (DC) method. Suspected isolates were examined by using mouse bioassay, PCR and real-time PCR methods. Clostridium botulinum type A and B strains were detected in 5 (2%) of 240 examined honey samples. Strains were also detected which were phenotypically similar to C. botulinum but which did not exhibit the ability to produce botulinum toxins and did not show presence of botulinum cluster (ntnh and bont genes) or expression of ntnh gene. The methods used in the examination, especially expression analysis of ntnh gene, enabled specific analysis of suspected strains and could be used routinely in environmental isolate analyses of C. botulinum occurrence.