Primers and probes for detection of toxin-producing (toxigenic) strains of Clostridium difficile, and to methods of detecting toxigenic strains using these primers and probes. Toxigenic strains of C. difficile are detected by nucleic acid-based amplification methods using particular primers and probes that bind to the toxin B (TcdB) gene. These primers and probes are used to amplify C. difficile nucleic acids in clinical samples to determine the presence of these toxigenic strains.