A multiplex PCR method has been developed to differentiate between the most common clinical serotypes of Salmonella enterica subsp. enterica encountered in Washington State and the United States in general. Six genetic loci from S. enterica serovar Typhimurium and four from S. enterica serovar Typhi were used to create an assay consisting of two five-plex PCRs. The assays gave reproducible results with 30 different serotypes that represent the most common clinical isolates of S. enterica subsp. enterica. Of these, 22 serotypes gave unique amplification patterns compared with each other and the other 8 serotypes were grouped into four pairs. These were further resolved by two additional PCRs. We compared the data from PCR serotyping with conventional serotyping and found that PCR serotyping was nearly as discriminatory as conventional serotyping was. The results from a blind test screening 111 clinical isolates revealed that 97% were correctly identified using the multiplex PCR assay. The assay can be easily performed on multiple samples with final results in less than 5 h and, in conjunction with pulsed-field gel electrophoresis, forms a very robust test method for the molecular subtyping of Salmonella enterica subsp. enterica.