The Gram-positive bacterium Erysipelothrix rhusiopathiae is a zoonotic pathogen that causes erysipelas in a wide range of mammalian and avian species. Historically, E. rhusiopathiae has been differentiated from other Erysipelothrix species by serotyping. Among 28 serovars of Erysipelothrix species, specific serovars, namely, 1a, 1b, and 2 of E. rhusiopathiae, are associated mainly with the disease in pigs, poultry, and humans; however, other serovar strains are often simultaneously isolated from diseased and healthy animals, indicating the importance of isolate serotyping for epidemiology. The traditional serotyping protocol, which uses heat-stable peptidoglycan antigens and type-specific rabbit antisera in an agar-gel precipitation test, is time-consuming and labor-intensive. To develop a rapid serotyping scheme, we analyzed sequences of the 12- to 22-kb chromosomal region, which corresponds to the genetic region responsible for virulence of serovar 1a and 2 strains of E. rhusiopathiae, of the 28 serovars of Erysipelothrix species. We confirmed that the serovar 13 strain lacks the genomic region and that some serovar strains possess very similar or the same genetic structure, prohibiting differentiation of the serovars. We created 4 multiplex PCR sets allowing the simultaneous detection and differentiation of the majority of Erysipelothrix serovars. Together with a previously reported multiplex PCR that can differentiate serovars 1a, 1b, 2, and 5, the multiplex PCR-based assay developed in this study covers all but one (serovar 13) of the reported serovars of Erysipelothrix species and should be a valuable tool for etiological as well as epidemiological studies of Erysipelothrix infections.