Serratia marcescens has been increasingly identified as a cause of infection in the immunocompromised host and in high-mortality-rate nosocomial outbreaks. It is thus important to use identification methods that allow study of the dynamics and evolution of nosocomial S. marcescens strains. The aim of this study was to identify S. marcescens strains isolated from nosocomial outbreaks in two pediatric hospitals by random amplification polymorphic DNA polymerase chain reaction (RAPD-PCR). RAPD-PCR was used to study five S. marcescens populations isolated from four different nosocomial outbreaks that occurred in two pediatric hospitals. This method was compared with the widely used biotyping system described by Grimont and Grimont. The combination of biotypification and RAPD-PCR allowed accurate identification of S. marcescens strains isolated in nosocomial outbreaks at pediatric hospitals; by RAPD-PCR, we were able to analyze clonal variations in S. marcescens populations. We established bacterial dissemination patterns in hospital environments according to hospital administration of medical services and compared changes in bacterial DNA amplification patterns in each hospital related with clonal variations by selective pressures. RAPD-PCR is a useful method to identify S. marcescens strains associated with nosocomial outbreaks.