Staphylococcus aureus is an opportunistic bacterial pathogen associated with asymptomatic colonization of the skin and mucosal surfaces of normal humans. Staphylococcus aureus isolates are often multidrug resistant. Rifampicin is a valuable antibiotic for Staphylococcal infection and it is effective in combination therapy, especially for deepseated infections, owing to its excellent pharmacokinetic properties and bactericidal activity. Rifampicin resistance, however, is frequent among methicillin-resistant Staphylococcus aureus (MRSA) in several countries. Prevalence of this bacterium was about 52% in patients with diabetic mellitus. Mutations in the rifampin resistance-determining (Rif) regions of Staphylococcus aureus were studied by gene amplification and sequencing methods. Five mutational changes were recorded in cluster I, II and III in rpoB gene of rifampicin resistant Staphylococcus aureus.