Background: Methods: This retrospective study enrolled CDI patients hospitalized between June 2023 and May 2024 at a large tertiary hospital in Southwest China, who were diagnosed via glutamate dehydrogenase (GDH) combined with toxin A/B testing. Toxigenic isolates from positive stool samples were submitted for antimicrobial susceptibility testing and whole-genome sequencing (WGS). Multilocus sequence typing (ST), identification of resistance determinants, and core-genome SNP (cgSNP) analysis were integrated with clinical data. Results: One hundred fifty-seven CDI patients were identified among 2,917 suspected patients with diarrhea. 67.5% of patients were male, 51.0% were ≥65 years, 20.4% had severe CDI, and two patients were fulminant. All 109 isolates remained susceptible to vancomycin and fidaxomicin. The moxifloxacin resistance rate reached as high as 56.0%, primarily driven by Conclusion: In the post-pandemic era, the clinical burden of CDI in Southwest China cannot be overlooked. ST81 with high-level fluoroquinolone resistance has increased significantly and deserves more attention. Integration of data on clinical cases and their pathogenic strains through sustained clinical case monitoring, genomic surveillance of isolates, and antimicrobial resistance (AMR) pattern surveys provides early warning for future clonal dissemination and supports clinical management and public health decisions.