Original Scientific Article Antimicrobial resistance (AMR) in Staphylococcus spp. is a growing problem in small animal practice, driven by the emergence of methicillin-resistant (MR) and multidrug-resistant (MDR) strains. This study analyzed 170 clinical Staphylococcus isolates from dogs in North Macedonia, using MALDI-TOF MS identification, disc diffusion susceptibility testing, and molecular detection of resistance genes (mecA, mecC, and blaZ). Staphylococcus pseudintermedius was identified as the most prevalent species (90%), followed by S. aureus (7.6%), S. hemolyticus (1.2%), S. schleiferi (0.6%), and S. intermedius (0.6%). Methicillin resistance was detected in 28.8% of the isolates by detecting mecA. Importantly, there was a significant discrepancy between phenotypic oxacillin resistance and mecA-positive isolates in S. pseudintermedius. Among the 49 mecA-negative but oxacillin-resistant isolates tested for blaZ, 65.3% were blaZ-positive, underscoring the critical role of beta-lactamase-mediated resistance. Overall, MDR was detected in 70.5% of isolates. High resistance was observed to multiple antibiotics, including penicillin G (73%) and clindamycin (61.8%), as well as critically important antibiotics (CIAs), such as fluoroquinolones, with resistance rates of 32.3% for enrofloxacin and 31.2% for marbofloxacin. Pradofloxacin showed the lowest resistance rate (22.3%). This study highlights the high prevalence of antimicrobial resistance in Staphylococcus spp. in dogs. Implementation of antimicrobial stewardship programs is critical to maintain the efficacy of key antimicrobials and ensure optimal treatment outcomes for companion animals in North Macedonia. https://macvetrev.mk/LoadArticlePdf/412 2024-04-30 i xiv https://doi.org/10.2478/macvetrev-2025-0022 Staphylococcus pseudintermedius companion animals methicillin resistance beta-lactam resistance multi-drug resistance Ivana Shikoska false 1 Faculty of Veterinary Medicine-Skopje, Ss. Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Zagorka Popova Hristovska false 2 Faculty of Veterinary Medicine-Skopje, Ss. Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Ivan Matevski false 3 Faculty of Veterinary Medicine-Skopje, Ss. Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Maja Jurhar Pavlova false 4 Faculty of Medicine, Ss. Cyril and Methodius University in Skopje, 50 Divizija No. 6, 1000 Skopje, North Macedonia AUTHOR Marija Ratkova Manovska false 5 Faculty of Veterinary Medicine-Skopje, Ss. Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Aleksandar Cvetkovikj false 6 Faculty of Medicine, Ss. Cyril and Methodius University in Skopje, 50 Divizija No. 6, 1000 Skopje, North Macedonia AUTHOR Iskra Cvetkovikj false 7 Faculty of Medicine, Ss. Cyril and Methodius University in Skopje, 50 Divizija No. 6, 1000 Skopje, North Macedonia AUTHOR Pomba, C., Rantala, M., Greko, C., Baptiste, K.E., Catry, B., van Duijkeren, E., et al. (2017). Public health risk of antimicrobial resistance transfer from companion animals. J Antimicrob Chemother. 72(4): 957-968.PMid:279990662. 1 https://doi.org/10.1093/jac/dkw481 Lord, J., Millis, N., Jones, R.D., Johnson, B., Kania, S.A., Odoi, A. (2022). 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