ABSTRACT Mupirocin is a topical antimicrobial used to eradicate methicillin-resistant Staphylococcus aureus (MRSA) colonization, usually in the absence of susceptibility testing. We hypothesized that high-level (HL) mupirocin resistance was associated with multidrug resistance (MDR). To this end, unique patient isolates identified at our institution during 2008 were stratified into those resistant to ≥3 non-β-lactam antimicrobial classes (MDR) and non-MDR MRSA. HL mupirocin resistance was screened by mupA PCR on all MDR isolates ( n = 191) and a 20% random sample ( n = 130) of non-MDR isolates; E-testing confirmed HL resistance. We found that among MDR isolates, 13 (6.8%) carried mupA , whereas none of the non-MDR isolates did ( P = 0.001). Thus, although the overall prevalence of HL mupirocin resistance is low among MRSA isolates at our institution, an association exists between mupA carriage and MDR. Using genotyping and antimicrobial susceptibility profiling, we identified nine HL mupirocin-resistant clones. Whereas the majority of mupA -negative MDR isolates had a health care-associated MRSA (HA-MRSA) genotype (multilocus sequence type 5 [ST5] or SCC mec type II), the majority of mupA -positive MDR isolates had a community-associated MRSA (CA-MRSA) genotype (ST8 or SCC mec type IV). However, CA- and HA-MRSA genotypes were more evenly distributed among mupA -positive isolates compared to mupA -negative MDR isolates. Thus, in Chicago, mupA is circulating among both CA- and HA-MRSA backgrounds.