Accurate and rapid detection of S. aureus is crucial to control its infections and minimize its leakage to the food chain. Therefore, the primary purpose of this research is to isolate S. aureus and characterize the isolates by coagulase production from cloacal, tracheal and nasal swabs of apparently healthy broiler & layer chickens and from different organs (joints, liver, lungs, ovaries, foot abscesses and heart blood) of diseased broiler & layer chickens. A total of 318 samples were collected as follows: 108 sample from different farms of apparently healthy broiler & layer and 210 samples from diseased layer & broiler chickens. The samples were examined bacteriologically, 164 (51.6%) Staphylococcal isolates were recovered from the 318 sample. 37/164 (22.6%) coagulase positive S. aureus were isolated. Upon confirmation by Real Time PCR for thermonuclease (nuc) gene using SYBR Green, isolates showed amplification at CT value 19, 20 and 21 with Melting temperature (Tm) values in the range of 76–77 oC. Dissociation curve between 75-80oC which confirmed as S. aureus. The isolated S. aureus was highly sensitive to vancomycin, amoxicillin + clavulinic acid and cephalothin by 84.5%, 83.8% and 78.4 respectively and highly resistant to ampicillin, oxacillin and penicillin by 75.7%, 73% and 70.2% respectively