ABSTRACT As clinical Salmonella enterica serovar Thompson (S. Thompson) emerged among the top ten prevalent serovars in China, understanding the distribution and origin of its multidrug-resistant (MDR) strains becomes imperative. This study employed antimicrobial susceptibility testing, whole-genome sequencing, and bioinformatics analysis to investigate the prevalence and genomic profiles of clinically important S. Thompson ST26 across China from 1997 to 2020. Upon analyzing 141 isolates from various sources, we identified 29 isolates, derived from 25 diarrhea patients and four animal-derived foods, displayed co-resistance to ciprofloxacin, cefotaxime, and azithromycin (CIPRCTXRAZIR), all of which are considered the front-line and critically-important antimicrobial agents for treating Salmonella infections in humans. The IncC plasmid was the predominant mobile vector identified among the CIPRCTXRAZIR isolates, harboring four crucial resistance genes qnrS1, qepA4, blaCMY-2, and mph(A) that confer resistance to three critically important antimicrobials. However, the closely related and clustered IncC-harboring CIPRCTXRAZIR isolates (0–23 single nucleotide polymorphisms [SNPs]) indicated the clonal spreading of these clinically important isolates in different provinces of China. Notably, the CIPRCTXRAZIR isolates appeared in aquatic products of animal-derived food, highlighting the possibility of aquaculture practices in the emergence and transmission of important antimicrobial resistance. Our findings emphasize the critical public health implications of IncC-carrying clinically important S. Thompson ST26. The study calls for enhanced surveillance of the clinically important S. Thompson ST26 clone in clinical and aquaculture and implementation of targeted interventions to mitigate its spread, thereby protecting food safety and public health.IMPORTANCEWe highlighted the critical veterinary public health issue of clinically important Salmonella enterica serovar Thompson (S. Thompson) prevalence in animal-derived foods, particularly aquatic products, calling for urgent action. The ability of S. Thompson to resist critically important antimicrobials across diverse environments highlights the transmission and survival of resistant strains within the livestock and poultry industry, aquaculture, and food production chains. This study underscores the importance of continuous surveillance of clinically important S. Thompson, especially in aquaculture settings, and considers the global trade of aquatic products as a potential vector for international dissemination. Further investigation on the factors contributing to the clone spread of clinically important Salmonella strain and the development of intervention strategies to mitigate its public health impact.