Background: Mupirocin is a topical antibiotic used for nasal decolonisation of methicillin resistant Staphylococcus aureus (MRSA). While resistance to mupirocin has been described it is usually not tested for in most clinical laboratories. Aim: The present study was carried out to detect the occurrence of mupirocin resistance in clinical isolates of Staphylococcus aureus in a tertiary care hospital set up in northern India. Materials and Methods: Staphylococcus aureus isolates obtained from clinical samples received in the microbiology laboratory over a period of one year were included in this study. Mupirocin resistance was detected by three phenotypic methods; disk diffusion method using 5μg and 200μg mupirocin disk to determine low-level and high-level resistance, broth microdilution method and an agar dilution method for determining minimum inhibitory concentrations. Methicillin sensitivity was also determined in the study isolates. Results: Of 250 non-duplicate Staphylococcus aureus isolates obtained, 5 (2%) were found resistant to mupirocin. All mupirocin resistance isolates were shown to have high-level resistance (minimum inhibitory concentration > 512μg/ml). All mupirocin resistant isolates were also resistant to methicillin. Results obtained by all three phenotypic methods showed good correlation. Conclusion: High-level mupirocin resistance is present in the northern Indian population which may be of major concern to prevent the spread of MRSA in hospitals and community.