Introduction: Prolonged and improper use of antibiotics increases the resistance among pathogens and leads to life threatening implications and increases mortality. The incidence of mupirocin resistance in Methicillin Resistant Staphylococcus aureus (MRSA) clinical isolates were reportedly increasing. Aim: To determine the rate of high level and low level mupirocin resistance in clinical MRSA isolates in a tertiary care hospital. Materials and Methods: A cross-sectional study was carried out for a period of three months from December 2019- February 2020 in Department of Microbiology, PSG Institute of Medical Sciences and Research (PSG IMSR), Coimbatore, Tamil Nadu, India. A total of 100 non duplicate Staphylococcus aureus isolates from different specimens were subjected to mupirocin susceptibility by Kirby-Bauer disc diffusion method as per Clinical and Laboratory Standards Institute (CLSI) 2019 standards. The low (MuL) and high (MuH) level mupirocin resistance were detected by using 5 and 200 μg mupirocin discs (Himedia) respectively. The isolate exhibiting diameter of ≥14 mm indicates its susceptibility. The isolate exhibiting diameter ≤13 mm for both 5 and 200 μg indicates MuL and MuH strains respectively. Pearson’s Chi-square test calculated, p-value of <0.05 was considered as statistically significant. Results: About 51 out of 100 (51%) Staphylococcus aureus isolates were found to be MRSA. In present study, 6 out of 51 (11.7 %) MRSA isolates were found to exhibit MuL and 5 out of 51 (9.8%) MRSA isolates were found to be having MuH. Low level and high level mupirocin resistance were not observed in Methicillin Sensitive Staphylococcus aureus (MSSA). Conclusion: The incidence of MuH and MuL resistance among MRSA were found to be 9.8% and 11.7% respectively. Screening for mupirocin resistant MRSA to be carried out periodically and stringent infection control practices to be in place to prevent further spread of mupirocin resistant MRSA.