Sixty percent to 80% of Clostridium difficile isolates in Korea have been reported to be toxigenic. However, over 1 year, we encountered a high number of tcdA-tcB+ strains associated with pseudomembranous colitis (PMC). C. difficile was isolated from 224 of 471 specimens (47.6%) from 371 patients. A subset of the culture-positive specimens (n = 106), containing no duplicate cases, was randomly selected for tcdA and tcdB polymerase chain reaction (PCR) assays. PCR results showed that tcdA+tcdB+ and tcdA-tcdB+ strains accounted for 39.6% (42/106) and 50.9% (54/106), respectively. Endoscopy, performed on 55/106 patients, revealed 29 with PMC, 5 with colitis, 14 with other colon diseases, and 7 normal cases. Among the 29 PMC cases, 21 (72.4%) were associated with tcdA-tcdB+ strains (P = 0.0016). These results revealed the possible emergence of tcdA-tcdB+ C. difficile strains in Korea, and these variant strains could evoke a higher rate of PMC than tcdA+tcdB+ strains.