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Objectives: In order to elaborate a new national challenge panel of resistant Gram-negative bacilli and Gram-positive cocci strains for the validation of routine antimicrobial susceptibility testing (AST) methods, an interlaboratory evaluation was organised. Methods: The results of 12 well-characterised multidrug-resistant strains tested by nine laboratories using local disk diffusion (DD) and automated AST (AUST) methods were compared with the reference broth microdilution method. Results: Overall categorical agreement ranged from 70% to 100% both for DD and AUST and was >90% for all but one strain for all antibiotics. Conclusion: Our multicentre AST study showed good reproducibility and the panel can be used as national resistant reference strains for routine AST validation. (C) 2022 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy. The Belgian national reference centre is supported in part by the Belgian Ministry of Social Affairs through a fund within the national health insurance system (INAMI-RIZIV). The authors thank the members of the Belgian National Antimicrobial Susceptibility Testing Committee (NAC): Olivier Denis, Stefanie Desmet, Youri Glupczynski, Marie Hallin, Te-Din Huang, Bénédicte Lissoir, Koen Magerman, Veerle Matheeussen, Pierrette Melin, Karl Mertens, Hector Rodriguez, Anne-Marie Van Den Abeele and Kris Vernelen. The authors also thank Audric Deckers for his help in compiling the statistics for this article.