Background. Methicillin-resistant Staphylococcus aureus (MRSA) pulsed-field type (PFT) USA300 causes skin and soft tissue infections in military recruits and invasive disease in hospitals. Chlorhexidine gluconate (CHG) is used to reduce MRSA colonization and infection. The impact of CHG on the molecular epidemiology of MRSA is not known. Objective. To evaluate the impact of 2% CHG-impregnated cloths on the molecular epidemiology of MRSA colonization. Design. Cluster-randomized, double-blind, controlled trial. Setting. Marine Officer Candidate School, Quantico, Virginia, in 2007. Participants. Military recruits. Intervention. Thrice-weekly application of CHG-impregnated or control (Comfort Bath; Sage) cloths over the entire body. Measurements. Baseline and serial (every 2 weeks) nasal and/or axillary swab samples were assessed for MRSA colonization. Molecular analysis was performed with pulsed-field gel electrophoresis. Results. During training, 77 subjects (4.9%) acquired MRSA, 26 (3.3%) in the CHG group and 51 (6.5%) in the control group (P = .004). When analyzed for PFT, 24 subjects (3.1%) in the control group but only 6 subjects (0.8%) in the CHG group (P = .001) had USA300. Of the 167 colonizing isolates recovered from 77 subjects, 99 were recovered from the control group, including USA300 (40.4%), USA800 (38.4%), USA1000 (12.1%), and USA100 (6.1%), and 68 were recovered from the CHG group, including USA800 (51.5%), USA100 (23.5%), and USA300 (13.2%). Conclusions. CHG decreased the transmission of MRSA-more specifically, USA300-among military recruits. In addition, USA300 and USA800 outcompeted other MRSA PFTs at incident colonization. Future studies should evaluate the broad-based use of CHG to decrease transmission of USA300 in hospital settings.