Abstract: Objective To investigate the pathogen spectrum of acute respiratory infections in Beijing during autumn and winter of 2023 to 2024. The findings will provide valuable insights for clinical diagnosis, treatment, prevention and control of respiratory infectious diseases through epidemiological analysis. Methods This study focused on patients who visited Peking Union Medical College Hospital for acute respiratory infections from September 2023 to February 2024 by convenience sampling.We used fluorescence quantitative PCR to detect six respiratory pathogens, namely influenza A virus (FluA), influenza B virus (FluB), Human rhinovirus (HRV), Mycoplasma pneumoniae (MP), respiratory syncytial virus (RSV), and adenovirus (ADV), in 5556 respiratory tract samples collected from patients with respiratory tract infections. Additionally, we analyzed the distribution of pathogen infections by combining the results with COVID-19 infections during the same period. Results Among the 5556 samples, the overall positive rate was 63.7%. The positive rates for single pathogen infections, dual pathogen infections, and infections with three or more pathogens were 54.0%, 8.9%, and 0.7% respectively. In terms of overall detection of pathogens, FluA and RSV were the predominant pathogens, with positive rates of 16.1% and 15.7% respectively. The positive rates for ADV, MP, HRV, and FluB were similar, at 11.1%, 11.1%, 10.0%, and 10.0% respectively. Although there was no difference in the overall pathogen positive rate between different genders and seasons, statistical differences were witnessed in the infection rates and pathogen compositions among different age groups (χ²=34.617, P<0.001, χ²=422.38, P<0.001). There were variations in the detection rates of MP, FluA, FluB, and HRV between different seasons (P<0.005). The positive rates of MP（χ²=8.647, P=0.003）、 FluA （χ²=131.932, P<0.001）和 HRV（χ²=174.199, P<0.001） were significantly higher in autumn than in winter, while FluB showed the opposite trend （χ²=287.894, P<0.001）, predominantly occurring in winter. The positive rates of MP, RSV, HRV, and ADV in the pediatric group were significantly higher than those in the middleaged and elderly patients (all P <0.001), while the positive rate of FluB in middleaged patients was significantly higher than that in both children and elderly patients (P <0.001 for both). The overall number of cases infected with the six respiratory pathogens decreased significantly during the period of COVID-19 infection, exhibiting an asynchronous seasonal pattern. Conclusion The detection rate of respiratory pathogens in Beijing was related to age and season. Targeted prevention and control measures should be implemented for different populations in different seasons, and early epidemic warnings should be issued.