Staphylococcus pseudintermedius represents one of the most frequently bacteria isolated on dog's skin and it was recently recognized as a zoonotic pathogen responsible for severe diseases also in humans. This study aimed to define the occurrence of canine methicillin-resistant and methicillin-susceptible S. pseudintermedius (MRSP and MSSP) strains and to compare their antimicrobial profiles. The study was carried out at veterinary microbiology laboratories of two different Italian veterinary teaching hospitals, Milan and Naples, from 2015 to 2017. The statistical comparison of the results revealed significant differences in MRSP occurrence (p-value = 0.0435) and MRSP and MSSP antibiotic resistance profiles. In Milan, MRSP strains displayed significantly higher antibiotic resistance percentages (p < 0.001) for some antibiotics, such as ceftriaxone and tobramycin, compared to those of Naples. Conversely, MSSP strains of Naples presented significantly higher rates (p < 0.001) of resistance to amoxicillin/clavulanic acid, kanamycin, erythromycin, and tetracycline than to Milan isolates. In conclusion, the results highlighted relevant variances among region-specific antibiotic resistance profiles, probably due to different antimicrobial selection pressures. Therefore, this study stands out the need for continuous monitoring of both MRSP and MSSP linked to different geographical areas, also considering their impact and importance on animal and human health.