We employed a multiplex polymerase chain reaction (PCR) coupled with capillary electrophoresis (mPCR-CE) targeting six Clostridium difficile genes,including tpi,tcdA,tcdB,cdtA,cdtB,and a deletion in tcdC for simultaneous detection and characterization of toxigenic C.difficile directly from fecal specimens.The mPCR-CE had a limit of detection of 10 colony-forming units per reaction with no cross-reactions with other related bacterial genes.Clinical validation was performed on 354 consecutively collected stool specimens from patients with suspected C.difficile infection and 45 isolates.The results were compared with a reference standard combined with BD MAX Cdiff,real-time cell analysis assay (RTCA),and mPCR-CE.The toxigenic C.difficile species were detected in 36 isolates and 45 stool specimens by the mPCR-CE,which provided a positive rate of 20.3％ (81/399).The mPCR-CE had a specificity of 97.2％ and a sensitivity of 96.0％,which was higher than RTCA (x2 =5.67,P =0.017) but lower than BD MAX Cdiff (P =0.245).Among the 45 strains,44 (97.8％) were determined as nonribotype 027 by the mPCR-CE,which was fully agreed with PCR ribotyping.Even though ribotypes 017 (n =8,17.8％),001 (n =6,13.3％),and 012 (n =7,15.6％) were predominant in this region,ribotype 027 was an important genotype monitored routinely.The mPCR-CE provided an alteative diagnosis tool for the simultaneous detection of toxigenic C.difficile in stool and potentially differentiated between RT027 and non-RT027.