Abstract: Objective To evaluate the detection specificity for clinical samples and the detection capability for standard substances of five commercially available multiplex polymerase chain reaction (PCR) nucleic acid detection kits (hereinafter referred to as the kits) for respiratory pathogens, and to provide a reference for selecting appropriate detection kits for multi-pathogen nucleic acid testing of respiratory infections. Methods Sixty known respiratory pathogen-positive clinical samples were selected and tested using the five kits (labeled A, B, C, D, and E). The detection rates and Kappa coefficients were calculated to evaluate the consistency between the results from these kits and those from single-pathogen PCR kits. According to the Limit of Detection (LOD) provided by the kits, standard substances of respiratory pathogens (including 12 types such as influenza virus, Mycoplasma pneumoniae, and Bordetella pertussis) were diluted to four concentrations (250, 500, 1000, and 2000 copies·mL⁻¹). All five kits were used for detection to evaluate their respective detection capabilities. Results Compared with the results from single-pathogen PCR kits, the five tested kits demonstrated good consistency (all Kappa > 0.8). Among them, Kit A had the highest detection rate (100%), followed by Kits C and E (98.33%), and then Kits B and D (95.00%). All five kits showed a relatively low false negative rate (FNR) for samples with a cycle threshold (Ct value) ≤ 35 (≤2.38%); however, for samples with Ct values > 35, the FNR increased (average FNR = 6.67%, P = 0.029). Kit C exhibited the highest detection sensitivity for the tested standard substances (average LOD: 458.33 copies·mL⁻¹), followed by Kit D, then Kits A/E (which were comparable and superior to the next), and finally Kit B. Conclusion The five multiplex PCR kits showed good consistency with single-pathogen detection results, but each had its own performance emphasis. Kit A, with the highest detection rate and high throughput, is suitable for targeted viral screening. Kit B, covering the broadest pathogen spectrum (including fungi/bacteria), is suitable for comprehensive respiratory pathogen screening. Kits C、 D and E, is applicable for rapid detection. It is important to note that the detection efficacy of all kits decreases for samples with low viral load (Ct value > 35). In practical application, selection should be based on specific screening objectives, throughput requirements, and sample types.