The incidence of Clostridioides difficile infection and the prevalence of hypervirulent ST1 (BI/NAP1/027)strain are increasing, especially in developing countries. We aimed to develop a new PCR assay for the identification of hypervirulent ST1 strains and toxigenic C. difficile in stool samples.We established a quadruplex TaqMan real-time PCR (pilW_4-plex PCR) assay targeting the pilW, a ST1-specific type Ⅳ minor pilin gene, and three C. difficile genes including cdtB, tcdB, and hsp. The sensitivity and specificity of the assay was tested using 403C. difficile isolates and 180 unformed stool sample. The results were compared with anaerobic culture-based conventional PCR method and MLST.The pilW_4-plex PCR identified toxigenic C. difficile in 333 (82.6%, 333/403) isolates with 100% sensitivity and specificity, and in 78 (43.3%, 78/180) stool samples with the sensitivity and specificity of 94.7% and 93.3%, respectively. Hypervirulent ST1 were detected in 21 strains and nine stool samples by the pilW_4-plex PCR. The pilW_4-plex PCR assay has no cross-reaction with non-toxigenic C. difficile or other bacteria.The pilW_4-plex PCR assay is an accurate and rapid method with high sensitivity and specificity for identification of ST1 and detection of toxigenic C. difficile in stool.Copyright © 2024. Published by Elsevier B.V.