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The present study aimed at examining the nasal and ear carriage of Staphylococcus aureus of Rwandan dogs and cats. Sixty-five S. aureus isolates were detected, all originating from the nostrils of dogs. Resistance to penicillin, penicillin/erythromycin/clindamycin, penicillin/tetracycline, and tetracycline solely was observed. The isolates were assigned to 23 different spa types, among them three novel (t21589, t21661, and t21662) variants, associated with eleven clonal complexes (CCs) (CC1, CC5, CC12, CC15, CC22, CC30, CC45, CC97, CC152, CC707, and CC834). Four isolates could not be assigned to any known CC. MLST revealed that one of them belonged to the new sequence type (ST) 9069. Panton-Valentine leukocidin (PVL) genes (lukF-PV/lukS-PV), the bovine leukocidin genes (lukM/lukF-P83), the toxic shock syndrome toxin gene tst-1, and various virulence-associated genes were detected. These findings demonstrate the dogs are colonized with human lineages of S. aureus. Coupled with the limited availability of S. aureus data from human medicine in Rwanda underscores the importance of hygiene measures and supports the need for a rigorous One-Health Surveillance program of the companion animals-human interface. The present study aimed at examining the nasal and ear carriage of Staphylococcus aureus of Rwandan dogs and cats. Sixty-five S. aureus isolates were detected, all originating from the nostrils of dogs. Resistance to penicillin, penicillin/erythromycin/clindamycin, penicillin/tetracycline, and tetracycline solely was observed. The isolates were assigned to 23 different spa types, among them three novel (t21589, t21661, and t21662) variants, associated with eleven clonal complexes (CCs) (CC1, CC5, CC12, CC15, CC22, CC30, CC45, CC97, CC152, CC707, and CC834). Four isolates could not be assigned to any known CC. MLST revealed that one of them belonged to the new sequence type (ST) 9069. Panton-Valentine leukocidin (PVL) genes (lukF-PV/lukS-PV), the bovine leukocidin genes (lukM/lukF-P83), the toxic shock syndrome toxin gene tst-1, and various virulence-associated genes were detected. These findings demonstrate the dogs are colonized with human lineages of S. aureus. Coupled with the limited availability of S. aureus data from human medicine in Rwanda underscores the importance of hygiene measures and supports the need for a rigorous One-Health Surveillance program of the companion animals-human interface.© The Author(s) 2025. Published by Oxford University Press on behalf of Applied Microbiology International.