Aims: To investigate the prevalence of Clostridium difficile encountered during sewage treatment and in water sources into which treated effluent was directly or indirectly discharged. Methods and Results: Samples from wastewater treatment plants (WWTPs) and rivers were collected and then enriched for Cl. difficile. Each of the isolates was subjected to toxinotyping and DNA typing using ribotyping, in addition to pulse-field gel electrophoresis. Cl. difficile was isolated from 92% (108/117) of the raw sludge and 96% (106/110) of the anaerobic digested sludge samples from two Ontario WWTPs. The pathogen was recovered from 73% (43/59) of dewatered biosolids and effluent discharge, in addition to river sediments 39% (25/64). Ribotype 078 (commonly associated with Community Acquired infections) was recovered from raw sewage (19%; 21/108), digested sludge (8%; 8/106), biosolids (35%; 15/43) and river sediments (60%; 15/25). Conclusions: Clostridium difficile is commonly encountered in raw sewage and survives the wastewater treatment process. The pathogen can then be disseminated into the wider environment via effluent and land application of biosolids. Significance and Impact of the Study: The study has illustrated the wide distribution of toxigenic Cl. difficile in WWTPs and river sediments although the clinical significance still requires to be elucidated.