<sec><b>Objectives</b><p>Studies on <italic>Clostridium difficile</italic> are rare in Korea. We investigated the epidemiological characteristics of <italic>C. difficile</italic> isolates from patients with <italic>C. difficile</italic>-associated disease (CDAD) in Korea.</p></sec><sec><b>Methods</b><p>Multiplex polymerase chain reaction was performed to detect the presence of <italic>tcdA</italic> and <italic>tcdB</italic> toxin genes. Antimicrobial susceptibility test was carried out by the disk-dilution method. <italic>C. difficile</italic> strains were subtyped by automated repetitive-element palindromic PCR (rep-PCR).</p></sec><sec><b>Results</b><p>Among patients with CDAD, 73 (25.8%), 32 (11.3%), 32 (11.3%), and 26 (9.2%) suffered from pneumonia, cancer or neoplasm, diabetes, and colitis, respectively. Of all stool samples, 43 samples (15.2%) were positive for <italic>C. difficile</italic> strains. We observed two expression patterns of toxin genes: <italic>tcdA</italic>+/<italic>tcdB</italic>+ (86% isolates) and <italic>tcdA</italic>−/<italic>tcdB</italic>+ (14% isolates), with all isolates expressing <italic>tcdB</italic>. Furthermore, some isolates were resistant to clindamycin (65%), ampicillin (56%), and cefazolin (40%), but all were susceptible to vancomycin and metronidazole. The tested samples were classified into diverse clusters using automated rep-PCR.</p></sec><sec><b>Conclusion</b><p>Our findings revealed the characteristics and antibiotic resistance of <italic>C. difficile</italic> isolates from patients in Korea. The epidemiological data may provide valuable insight into development of treatment strategies for <italic>C. difficile</italic> infections in Korea.</p></sec>