Introduction: Staphylococcus aureus is one of the common colonizers of human andrnseveral animal species with the capacity to cause serious diseases. It is one of the globalrnpublic health priority pathogens due to its ubiquitous nature, ability to cause life-threateningrndiseases and continuous evolvement of drug resistance. The emergence and spread ofrnmultidrug resistant strains, mainly MRSA, initially in the health care setting, later in therncommunity and recently in the veterinary side with a capacity of transmission to human is ofrnspecial concern. Several molecular techniques have been used successfully for reliablernidentification, determination of specific virulence and drug resistance genes and tornunderstand spread and distribution of specific S. aureus strains. In Ethiopia, despite severalrnreports on phenotypic studies of S. aureus isolates from human and animals, data onrnmolecular characterizations is scarce. rnObjectives: To isolate S. aureus from human skin and soft tissue infections, milk of dairyrncows and nares of dairy farm workers in Mekele, Northern Ethiopia; and then to determinernthe antimicrobial resistance pattern, virulence genes profile and genotypic diversity andrnrelatedness of the isolates. rnMethods: A cross sectional study was conducted during period of March 2016 to Augustrn2017 G.C. A total of 811 non-duplicate specimens (355 wound/pus swabs from humanrnpatients with skin and soft tissue infections [SSTIs] attending Ayder referral hospital, 71rnnasal swabs from dairy farmers and 384 milk samples from dairy cows) were collected to rnisolates S. aureus. Culture and identification of S. aureus was performed using standardrnlaboratory procedures. Antimicrobial susceptibility testing was performed following thernCLSI guideline. All phenotypic characterizations of S. aureus were done at thernMicrobiology laboratory of Ayder Referral Hospital, Mekele, Ethiopia. All S. aureusrnisolates were stored at âˆ’70rn0rnC and shipped to USA for Molecular characterizations at thernInfectious Diseases Molecular Epidemiology Laboratory (IDEML) of the Ohio StaternUniversity and the Public Health Research Institute of the State University of New Jersey,rnUSA. Molecular characterizations include confirmation of S. aureus by nuc detection, rnmecA/mecC detection, toxin genes profiling (pvl, nine se genes and tsst-1), spa typing andrnSCCmec typing. rnResults: From the 811 samples, 193 (23.8%) were positive for S. aureus using phenotypicrnand molecular methods; where 123 (63.7%) were from human SSTIs, 22 (11.4%) from naresrnof dairy farmers and 48 (24.9%) from milk of Dairy cows. Overall, high percentage ofrnresistance was observed for Penicillin (92.7%); however, no resistance was found forrnVancomycin, Daptomycin and Rifampin. Four (2.1%) of the 193 isolates were MRSA wherernall were from human and were both cefoxitin resistant and mecA positive. Two of themrncarried SCCmec type III and the other two SCCmec type IV. Moreover, 26% of S. aureusrnisolates were multidrug resistant. Regarding toxin genes carriage, 66.8% of the total isolatesrnpossessed at least one of the 11 targeted toxin genes. Of the 11 genes, pvl was the mostrnfrequently detected (carried by 36.8%) followed by seg and sei (29% each), seb (19.2%), searn(16%) and sec (15%). Interestingly, pvl was found significantly higher (p