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Objectives: Clostridioides (previously Clostridium) difficile is a major growing cause of nosocomial diarrhoea known as C. difficile infection (CDI). This study investigated the prevalence and antimicrobial resistance patterns of C. difficile isolated from patients suffering from diarrhoea in Iran between 2016-2018., Methods: A total of 151 stool specimens were collected and were screened for the presence of C. difficile. Specimens were examined for toxins by culture, enzyme immunoassay (EIA) and PCR. Antimicrobial susceptibility testing was performed for 12 antibiotics (metronidazole, vancomycin, clindamycin, tetracycline, erythromycin, ciprofloxacin, levofloxacin, moxifloxacin, fusidic acid, piperacillin, piperacillin/tazobactam and rifampicin) by the disk diffusion method according to the guidelines of the CLSI, EUCAST and CA-SFM., Results: Of 151 stool specimens, 66 (43.7%) were positive for C. difficile by PCR, whereas 2 (1.3%) were only positive for C. difficile toxins based on EIA. A total of 292 clostridial isolates were obtained from specimens by culture, of which 133 (45.5%) were finally confirmed as C. difficile by PCR. Of 121 isolates resistant to at least one antibiotic, 107 (88.4%) were resistant to three or more antimicrobials and thus were defined as multidrug-resistant (MDR). Different and diverse resistance patterns to the antimicrobial drugs were seen among the isolates., Conclusion: This is the first report of the isolation of C. difficile from different governmental hospitals of Iran and indicates that CDI might be an important nosocomial infection in different hospital wards. Moreover, this study provides a comprehensive picture of the MDR phenotype characteristics of C. difficile isolates in Iran., (Copyright © 2019 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)