Abstract: OBJECTIVE To investigate the clone distribution and the antimicrobial susceptibility of the Clostridium difficile strains isolated from the patients diagnosed with hospital-acquired diarrhea in Hangzhou. METHODS Stool samples were collected from the patients who were diagnosed with hospital-acquired diarrhea and hospitalized the First Affiliated Hospital and Sir Run Run Shaw hospital, School of Medicine, Zhejiang University from Jan 2013 to Dec 2016, the C.difficile strains were cultured and screened, Species identification and toxin detection were then carried out by using PCR followed by sequencing on the target genes of 16-23S rRNA gene and toxin genes (tcdA and tcdB), respectively.The homology of toxin-producing strains was analyzed by means of MLST, and the drug susceptibility testing was performed for the C.difficile strains. RESULTS Of totally 61 strains of C.difficile cultured, 54 were positive for toxin genes, there were 1 strain (A+B-) positive for tcdA and 53 strains (A-B+)positive for tcdB; no strain harbored both tcdA and tcdB genes.Eleven different STs were identified among 54 toxin strains, 7 of which were reported previously.ST54 was the most popular epidemic clone type (38.9%, 21/54), followed by ST2, ST35 and ST37 (13%, 7/54).The result of drug susceptiblity testing showed that vancomycin and metronidazole remained the most sensitive antibiotics, the drug resistance rates to levofloxacin, tetracycline and ciprofloxacin were 16.6%, 31.4% and 38.8%, respectively, and the drug resistance rates to clindamycin and fusiacid were the highest (96.3%). CONCLUSION The ST54 type C.difficile strains that are positive for toxin phenotype A-B+ are the predominant clone type in the district.Vancomycin and metronidazole remain the most effective antibiotics for treatment of C.difficile infection.