Staphylococcus aureusis among the top three causative agents of nosocomial infection in Ethiopia. The majority of studies in Ethiopia have focused on the epidemiology ofS. aureusin hospital settings, with limited molecular genotyping results. Molecular characterization ofS. aureusis essential for identification of strains, and contributes to the control and prevention ofS. aureusinfection. The aim of the current study was to determine the molecular epidemiology of methicillin-susceptibleS. aureus(MSSA) and methicillin-resistantS. aureus(MRSA) isolates recovered from clinical specimens in Ethiopia. A total of 161 MSSA and 9 MRSA isolates were characterized using pulsed-field gel electrophoresis (PFGE) and staphylococcal protein A (spa) typing. Based on the PFGE analysis, MSSA isolates were grouped into eight pulso-types groups (from A to I), while MRSA isolates clustered into three (A, B and C) pulso-types with more than 80% similarity. Thespatyping analysis showed diversity ofS. aureuswith 56 distinctspatypes.Spatype t355 was most prevalent (56/170, 32.9%), while eleven newspatypes were detected including t20038, t20039, and t20042. The identifiedspatypes were clustered into 15spa-clonal complexes (spa-CCs) using BURP analysis; novel/unknownspatypes were further subjected to MLST analysis. The majority of isolates belonged tospa-CC 152 (62/170, 36.4%), followed byspa-CC 121 (19/170, 11.2%), andspa-CC 005 (18 /170, 10.6%). Of the nine MRSA isolates, 2 (22.2%) werespa-CC 239 with staphylococcal cassette chromosome (SCC)mecIII. These findings highlight the diversity ofS. aureusstrains in Ethiopia, as well as the presence of potentially epidemic strains circulating in the country necessitating further characterization ofS. aureusfor antimicrobial resistance detection and infection prevention purposes.