Abstract: OBJECTIVE To investigate the etiological characteristics of hospitalized children with diarrhea in a children's hospital of Wuhan from 2022 to 2025 and analyze the prevalence trend so as to provide bases for precise prevention and control. METHODS A total of 6688 children who were hospitalized in Wuhan Children's Hospital due to diarrhea-related diseases from Mar. 1, 2022 to Dec. 31, 2025 were recruited as the research subjects. The bacterial pathogens were identified by means of standard microbial culture combined with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis, and the viral pathogens were identified by colloidal gold immunochromatography for rotavirus/adenovirus and norovirus. RESULTS The pathogens were tested positive in 2009 cases, and totally 2248 strains of pathogens, including 1521 strains of viruses and 727 strains of bacteria and fungi, were isolated. Rotavirus (19.44%), norovirus (9.13%), Salmonella (7.78%) and adenovirus (5.43%) were the major pathogens. Totally 197 patients had mixed infections, accounting for 9.81% among the positive cases, and the patients with the mixed infections of virus-virus were dominant. The rotavirus infection was highly prevalent among the children with 3 to 6 years of age (33.03%), and the prevalence rate was 41.62% in spring, 18.87% in winter. Salmonella infection was highly prevalent among the infants less than 1 year old, and the prevalence rate was 12.58% in spring, 11.24% in autumn. Norovirus and adenovirus infections exhibited sporadic distribution throughout the year, with a higher incidence observed in the children with 1 to 6 years of age. The isolation rates of rotavirus, norovirus and adenovirus showed an annual increasing trend, whereas the isolation rate of Salmonella decreased annually; there were significant differences (all P＜ 0.05). CONCLUSIONS The viruses are dominant among the pathogens isolated from the children with diarrhea in Wuhan. Rotavirus and Salmonella are identified as the two core pathogens, exhibiting distinct ‘age-seasonal' epidemic patterns. It is recommended to strengthen the rotavirus vaccination for children with 1 to 6 years of age during winter and spring, intensify the regulatory oversight of food and drinking water safety during summer and autumn, and implement molecular diagnostics to enhance surveillance systems.