Background: Contagious equine metritis (CEM), caused by Taylorella equigenitalis, is a notifiable disease in many countries, responsible for reproductive disorders, making diagnosis essential before each reproduction season for mares and stallions. Klebsiella pneumoniae and Pseudomonas aeruginosa are also often identified in equine metritis, obliging laboratories to perform analysis for these 3 pathogens systematically. Bacteriology is the gold standard diagnostic method, and since 2024, PCR use has been expanded in several studbooks as in the HBLB Codes of Practice. Objectives: to evaluate the performance of a freeze-dried quadruplex qPCR kit(1) for detection of the 3 pathogens with a direct lysis protocol. Study design: assay validation Methods: Samples from the 2024 reproductive season were tested with the new kit and results were compared to those obtained with PCR and bacteriology usually used at LABEO. Limit of detection and exclusivity were measured for each pathogen. Subsequently, 292 DNA extracts from fresh AMIES-charcoal swabs were tested randomly among samples received at LABEO. Results: LDPCR was determined at 6.25, 75, and 6.25 gene copies/PCR for T. equigenitalis, K. pneumoniae, and P. aeruginosa respectively. For all three pathogens, measured sensitivity was 100% (n = 89; CI95: 95.9%–100%), with good exclusivity. Of 292 samples taken from LABEO, none was positive for T. equigenitalis and 1 sample was positive for P. aeruginosa with both PCR methods. For K. pneumoniae, 178 samples were positive with the PCR usually used at LABEO, while only 21 were positive on the new PCR and 4 were confirmed in bacteriology. Main limitations: Number of field positive samples for T. equigenitalis. Conclusions: The new PCR kit gives rapid results for three pathogens simultaneously and better correlation with bacteriology results regarding K. pneumoniae, which simplifies the workflow in laboratories while increasing the results' accuracy. Key manufacturer:1 Quadruplex qPCR kit, IDvet: www.innovativediagnostics. com. Ethical animal research: Not required: excess material from clinical samples was used. Informed consent: Not stated Competing interests: None declared. Funding: None.