Abstract: OBJECTIVE To analyze the epidemiological characteristics of non-bacterial pathogens in hospitalized children with acute respiratory tract infection (ARTI) across multiple centers in Fujian Province from 2023 to 2024. METHODS A retrospective cohort study was conducted, enrolling 51 078 hospitalized children with ARTI from 10 regional pediatric medical centers in Fujian between Jan. 2023 and Dec. 2024. Eleven non-bacterial respiratory pathogens were uniformly detected through multiplex polymerase chain reaction (PCR) based on Sanger sequencing platform fragment analysis technology, including influenza A virus (FluA), influenza B virus (FluB), parainfluenza virus (PIV), respiratory syncytial virus (RSV), adenovirus (ADV), human rhinovirus (HRV), human bocavirus (HBoV), human coronavirus (HCoV), human metapneumovirus (HMPV), Mycoplasma pneumoniae (Mp) and Chlamydia (Ch). Categorical data were described as n(%), with intergroup rate comparisons performed based on chi-square tests/Fisher's exact probability method. The correlation of positive detection rates and pathogen spectrums with age, gender and season was analyzed. RESULTS The overall detection rate increased from 2023 to 2024 (71.21% to 76.67%)), with a concurrent rise in mixed infection rates (12.62% to 14.96%) (all P<0.001). The top four pathogens shifted from HRV, Mp, RSV and FluA (2023) to HRV, Mp, ADV and RSV (2024). Age factors: The 3-<6 years group had the highest detection rate (78.80%), with significant differences among age groups and pathogen distributions (P<0.001). Gender differences: Detection rates were higher in boys than girls (P<0.05), with HRV, RSV and Ch significantly more prevalent in boys, while Mp and HMPV were more common in girls (P<0.05). Seasonal trends: All pathogens exhibited significant seasonal variations (P<0.001). HRV consistently peaked in autumn, Mp sharply declined after outbreak, RSV shifted from spring-summer peaks in 2023 to summer-autumn in 2024, and ADV surged in 2024. CONCLUSION The characteristics of non-bacterial pathogen spectrum in children with ARTI in Fujian from 2023 to 2024 are as follows: the overall detection rate exceeds 70%, correlating with age and gender, and demonstrating a distinct seasonal distribution trend.