Clostridium difficile is a bacterial enteric pathogen that can cause nosocomial diarrhea. Currently, the prevalence and severity of C. difficile infection have increased rapidly due to the overuse and misuse of antibiotics and the emergence of hypervirulent strains. The aims of this study were to determine the prevalence of C. difficile in hospitalized diarrhea patients admitted at Ramathibodi hospital during January 2010 – December 2010 and to characterize these isolates for the presence of toxin genes and their molecular type. C. difficile was detected from 52 of 216 (24%) patients stool samples. C. difficile isolates were recovered from C. difficile positive stool samples using selective culture method. A multiplex PCR method was used to analyze the C. difficile toxin genes including tcdA, tcdB, cdtA and cdtB. Strains identity were determined by a PCR-ribotyping method.  Four toxin type groups of strains were found among 24 C. difficile isolates which toxin type group tcdA⁺ tcdB⁺ cdtA/cdtB⁺ was a predominantly group of strains. Sixteen different PCR-ribotypes patterns were classified which ribotype 017 was the most common type comprised 29% of C. difficile strains occurring in the hospital.