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INTRODUCTION Clostridioides difficile is a major pathogen responsible for hospital-associated diarrhoea. This study investigated the molecular epidemiology and antibiotic resistance of C. difficile isolates in five Algerian hospitals. METHODOLOGY Between 2016 and 2019, faecal specimens were collected from in-patients and were cultured for C. difficile. Isolates were characterised by toxin genes detection, Polymerase Chain Reaction (PCR)-ribotyping, Multilocus Sequence Typing (MLST), antimicrobial susceptibility testing against a panel of antibiotics, and screened for antimicrobial resistance genes. RESULTS Out of 300 patient stools tested, 18 (6%) were positive for C. difficile by culture, and were found to belong to 11 different ribotypes (RT) and 12 sequence types (ST): RT 085/ST39, FR 248/ST259, FR 111/ST48, RT 017/ST37, RT 014/ST2, RT 014/ST14, FR 247/new ST, RT 005/ST6, RT 029/ST16, RT 039/ST26, RT 056/ST34 and RT 446/ST58. MLST analysis assigned the isolates to two clades, 1 and 4. Clade 4 was more homogeneous, as it mainly included non-toxigenic isolates. Three toxin gene profiles were detected, two toxigenic, A+B+CDT- (33.3%) and A-B+CDT- (11%); and one non-toxigenic, A-B-CDT- (55.5%). All C. difficile isolates were susceptible to metronidazole, vancomycin and moxifloxacin. CONCLUSIONS Overall prevalence of C. difficile in our healthcare settings was 6%. Antibiotic resistance rates ranged from 72.2% (clindamycin) to 16.6% (tetracycline). This study highlighted a relatively high genetic diversity in term of ribotypes, sequence types, toxin and antibiotic resistance patterns, in the C. difficile isolates. Further larger studies are needed to assess the true extent of C. difficile infections in Algeria.