Whether targeted or universal decolonization strategies for the control of methicillin-resistant Staphylococcus aureus (MRSA) select for resistance to decolonizing agents is unresolved. The REDUCE-MRSA trial (ClinicalTrials registration no. NCT00980980) provided an opportunity to investigate this question. REDUCE-MRSA was a 3-arm, cluster-randomized trial of either screening and isolation without decolonization, targeted decolonization with chlorhexidine and mupirocin, or universal decolonization without screening to prevent MRSA infection in intensive-care unit (ICU) patients. Isolates from the baseline and intervention periods were collected and tested for susceptibility to chlorhexidine gluconate (CHG) by microtiter dilution; mupirocin susceptibility was tested by Etest. The presence of the qacA or qacB gene was determined by PCR and DNA sequence analysis. A total of 3,173 isolates were analyzed; 2 were nonsusceptible to CHG (MICs, 8 μg/ml), and 5/814 (0.6%) carried qacA or qacB At baseline, 7.1% of MRSA isolates expressed low-level mupirocin resistance, 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 an 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 or qacB were rare among MRSA isolates in the REDUCE-MRSA trial. The odds of mupirocin resistance were no different in the intervention versus baseline periods across arms, but the confidence limits were broad, and the results should be interpreted with caution.