Background: Two types of mupirocin resistance among methicillin resistant Staphylococcus aureus (MRSA) have been reported; low-level mupirocin resistance (LL-MR), and high-level mupirocin resistance (HL-MR). The mupA gene is typically located on mobile genetic elements which facilitate the resistance dissemination. Objective: The aim of this work was to identify the mupA gene location, as well as the restriction fragment length polymorphism (RFLP) patterns in high and low-level mupirocin resistant MRSA. Methodology: This study was conducted on 100 MRSA isolates; seven of them were mupirocin resistant. The E test was used to identify high and low level mupirocin resistance. Amplification of mupA gene in total and plasmid DNA was performed. We also detected the spacer region (trsLM–IS257-like–mupA) in the 7 isolates by PCR then we investigated its RFLP patterns. Results: Four MR MRSA isolates had low level resistance, their MupA gene was located on chromosomal DNA, whereas, three isolates showed high level MR, their MupA gene was located on plasmid DNA. Four types of different RFLP patterns of the spacer region were identified; type-1 included two LL-MR isolates, each of type-2 and 3 included both HL-MR and LL-MR isolates, and type-4 included one HL-MR isolate. Conclusions: Staphylococcus aureus mupA gene responsible for LL-MR is located on the chromosome while that responsible for HL-MR is plasmid-mediated. The spacer region variations appear to occur in both chromosomal and plasmid-located mupA gene regardless the type of mupirocin resistance.