Whether targeted or universal decolonization strategies for control of methicillin-resistant Staphylococcus aureus (MRSA) select for resistance to decolonizing agents is unresolved. The REDUCE MRSA trial provided an opportunity to investigate this question. REDUCE-MRSA was a 3-arm, cluster-randomized trial of screening and isolation without decolonization, targeted decolonization with chlorhexidine and mupirocin, or universal decolonization without screening to prevent MRSA infection in intensive care unit patients. Iisolates from baseline and intervention periods were collected and tested for susceptibility to CHG by microtiter dilution; mupirocin susceptibility was tested by E-test. Presence of qacA/B genes was determined by PCR and DNA sequence analysis. A total of 3,173 isolates were analyzed; two were non-susceptible to CHG (MICs 8 μg/mL), and 5/814 (0.6%) carried qacA/B. At baseline, 7.1% of MRSA isolates expressed low-level and 7.5% expressed high-level mupirocin resistance. In a mixed effects generalized logistic regression model, the odds of mupirocin resistance among clinical MRSA isolates or MRSA isolates acquired in ICU in intervention versus baseline periods did not differ across arms, although estimates were imprecise due to small numbers. Reduced susceptibility to chlorhexidine and carriage of qacA/B were rare among MRSA isolates in the REDUCE MRSA trial. Odds of mupirocin-resistance were no different in the intervention versus baseline periods across arms, but confidence limits were broad and results should be interpreted with caution.