Abstract: OBJECTIVE To investigate the molecular characteristics of antimicrobial resistance genes, virulence genes, and mobile genetic elements (MGEs) in binary toxin-producing Clostridium difficile strains in Xi'an. METHODS The three strains of clinical ST5 C. difficile isolates (CD2019008, CD2022013 and CD2023025)were sequenced. The sequencing results were interpreted by Prokka software, the sequences at PaLoc and CdtLoc regions were analyzed by Easyfig software, the drug resistance gens and virulence genes were predicted by Abricate software; the antimicrobial susceptibilities of the three strains of C. difficile were detected by E-test strips, and the MGEs were observed with the use of ICEscreen, ISEScan, PlasmidFinder and phage search tools. RESULTS The genome sizes of the three strains of C. difficile ranged from 4.07 to 4.15 Mbp. The three strains of ST5 isolates carried larger PaLoc region than the RT027/ST1 isolates and RT078/ST11 isolates and were inserted with Tn6218 transposable element and encoded a truncated TcdC protein. They harbored complete binary toxin encoding and regulatory genes, which showed high homology with those in the RT027/ST1 strains. In addition to the toxin genes tcdA, tcdB, and cdtA/B, the strains carried 6 cell adhesion genes, 1 extracellular enzyme gene, and 5 antimicrobial resistance genes. All of the three strains of C. difficile were resistant to ciprofloxacin; the strain CD2019008 was resistant to levofloxacin but was sensitive to other antibiotics. ICEs and IMEs were identified in two strains of C. difficile, and the predominant family of inserted sequence (IS) was IS200/IS605. CONCLUSIONS The virulence and drug resistance gene spectrum, characteristics of MGEs and peculiar transposon Tn6218 are revealed in the study through the analysis of whole genomic characteristics of the three strains of binary toxin-producing C. difficile and comparison with the prevalent strains of type 027 and 028. The strain possesses the potential to emerge as a hypervirulent predominant strain, and it is necessary to strengthen the surveillance and study of ST C. difficile.