The present study was conducted to determine the molecular diversity of Staphylococcus aureus strains isolated from human, bovine and food samples based on the polymorphism of the spa gene. A total of 208 S. aureus isolated from human, bovine raw milk and food samples were assessed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and single locus sequence typing (SLST) methods, followed by determination of spa types using Ridom SpaServer. Altogether, 15 distinct RFLP patterns were recorded (I-XV). The highest heterogeneity was observed among S. aureus isolated from humans, whereas most of bovine and food S. aureus isolates indicated certain RFLP patterns. Although most of the isolates from patients showed RFLP pattern I, none of the S. aureus isolated from carriers had this spa pattern. Besides, the results of SLST led to the characterization of 16 spa types, and one of them was a novel spa type which has been registered in Ridom SpaServer for the first time and designated as type t16929. Determination of a high number of shared RFLP patterns between human and food S. aureus isolates indicated possible transmission of S. aureus and the source of food contamination. Thus, effective hygiene measures should be taken to break transmission routes. However, it seems that S. aureus isolated from patients, carriers and bovine should be considered in a different way, since some isolates had similar patterns, while the others showed their own specific pattern.