Clostridium difficile is the most common and important cause of toxigenic colitis in the health care setting. Laboratory diagnostics have included bacterial culture with further identification of toxigenic stains, or more commonly, direct detection of preformed toxin in stool samples using biological or immunochemistry assays. Recently, molecular amplification assays for the direct detection of toxin-encoding genes have become available commercially. We prospectively evaluated 2 FDA-cleared molecular amplification tests, the Illumigene C. difficile and the ProGastro Cd PCR assay, for the direct detection of toxigenic C. difficile from fecal samples. Of 446 samples tested, 418 produced matching amplification results, 88 positive and 330 negative, and 13 resolved with repeat testing. Toxigenic culture and direct cytotoxin testing were used to resolve the remaining 15 discordant samples. Overall, each assay performed well and correctly identified 97% of positive samples.