Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea worldwide. In order to gain a better understanding about the molecular epidemiology of C. difficile in Beijing, China, we performed molecular typing, antimicrobial susceptibility test, and drug resistance gene sequencing on 174 C. difficile strains collected from four large tertiary hospitals in Beijing. A total of 31 sequence types (STs) were identified among the 174 strains, of which ST81 was found to be the most prevalent clone, accounting for 26.4% (46/174) of the isolates, followed by ST2 (16.7%, 29/174) and ST54 (9.8%, 17/174). All the isolates were susceptible to metronidazole and vancomycin. The test strains displayed resistance rates of 97.1%, 44.3%, and 44.3% for ciprofloxacin, levofloxacin, and moxifloxacin, respectively. The ST81 isolates displayed a drug resistance rate of 97.8% for the antibiotics levofloxacin and moxifloxacin, which was significantly higher than the rates of the ST2 (0%), ST54 (17.6%), and ST42 (0%) isolates (P<0.05). An amino acid mutation, T82I, was identified in GyrA and the total mutation rate of the C. difficile strains was 40.8% (71/174), while the mutation rate of the ST81 strains was 95.7% (44/46). Three amino acid mutations including D426N, S366A, and D426V were identified in GyrB and the total mutation rate of GyrB was 39.1%. The double-site mutation in GyrB, S366A+D426V was identified in all the ST81 (n=46) strains. In conclusion, the C. difficile ST81 clone showed high levels of fluoroquinolone resistance in Beijing, China, thus highlighting the need for nationwide surveillance of CDI.