Background Although the epidemiology of Clostridioides difficile is important, few studies examining transmission of C. difficile have been reported, especially in wards with low detection rates, such as neurosurgery departments. Purpose This retrospective study investigated the epidemiology of C. difficile infection in a neurosurgery department over a 24-month period, particularly examining the transmission of C. difficile using whole-genome sequencing (WGS). Methods Clostridioides difficile strains were isolated and identified from fecal samples of neurosurgical patients. Toxigenic strains were typed using multilocus sequence typing, PCR ribotyping and using capillary gel electrophoresis. WGS was used to characterize C. difficile ST-37/RT017 isolates, and comparative genomic analyses were performed to compare genomic differences between all ST-37 strains from other wards. The susceptibility to 8 antimicrobial agents was examined using the E-test. Results Comparative genomic analyses revealed that isolates obtained from neurosurgical patients clustered into two lineages. Only strains s11052403 and s10090304, respectively, isolated from a patient on the 8th floor of the neurosurgery ward and a patient on the 9th floor, were highly similar, exhibiting differences of only two single-nucleotide polymorphisms. All C. difficile ST-37/RT017 strains isolated from neurosurgical patients were resistant to multiple classes of antibiotics. Conclusion There is an urgent need to raise awareness of C. difficile infection, and epidemiologic surveillance is required to detect clustering and transmission of C. difficile cases in China. Strict disinfection of the environment is essential to reduce transmission of C. difficile and achieve effective infection control in the hospital setting.