BACKGROUND: Laboratory detection methods are commonly tested against gold standards and corresponding reference methods to confirm their suitability and efficiency. OBJECTIVE: The IEH Listeria Test System (multiplex PCR) was tested against Health Canada's reference method (MFHPB-30) for the simultaneous detection of Listeria spp. and Listeria monocytogenes, on three different surfaces: plastic (PL), sealed concrete (SC) and stainless steel (SS). METHODS: The R2 medium was used for pre-enrichment of the samples at 35 °C for 24 h, followed by the IEH Listeria multiplex PCR. Each individual surface coupon (PL, SC, SS) was inoculated with either Listeria innocua, L. monocytogenes, or L. welshimeri along with two non-target microorganisms of concern and subjected to candidate (IEH Listeria multiplex PCR) and reference method (MFHPB-30) testing in an unpaired manner, and included culture confirmation for IEH method. RESULTS: The candidate method demonstrated 0% false negative, 0% false positive, 100% sensitivity, and 100% specificity after only 24 h enrichment followed by Multiplex PCR. CONCLUSION: Evaluating the differences between the candidate and reference method detection probabilities across all three surface types revealed that the candidate method was equivalent to MFHPB-30, while requiring significant less time for detection.