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The indiscriminate use of first-line drugs contributed to the spread of resistant bacteria, a major concern for both human and veterinary medicine. Methicillin resistance is acquired through the mecA gene, which encodes for the PBP2a protein and lends the resistance to β-lactams. Verifying the correspondence between gene harboring and protein expression and accelerating methicillin resistance diagnosis is critical to improve the management of antimicrobial administration and to reduce the spread of drug resistances. We tested the applicability of immunofluorescence targeting PBP2a protein to identify a new potential methicillin resistance screening test, ancillary to conventional culture methods. We collected 26 clinical Staphylococcus pseudintermedius (SP) isolates: 25 from canine pyoderma and 1 from dermatitis in a dog owner. SP is one of the most important etiological agents in canine pyoderma and can harbor the mecA gene. We performed PCR for mecA gene detection, broth microdilution (BMD) for phenotypic methicillin resistance, and immunofluorescence targeting PBP2a protein. Compared to the PCR as the gold standard, immunofluorescence showed an apparent prevalence of 34.6% vs. a true prevalence of 53.8%, with 100% specificity, 64.3% sensitivity, and 80.8% diagnostic accuracy. PBP2a expression showed isolate-dependent variability: in some isolates, most of the bacterial cells showed an intense and clearly membranous pattern, while in others only a few of them could be detected. Performing the assay in duplicate improved the diagnostic accuracy. Since the mecA gene is shared among the members of the …