Clostridium perfringens type A isolates carrying the enterotoxin (cpe) gene are important causes of both food poisoning and non-food-borne diarrheas in humans. In North America and Europe, food poisoning isolates were previously shown to carry a chromosomal cpe gene, while non-food-borne gastrointestinal (GI) disease isolates from those two geographic locations were found to have a plasmid cpe gene. In this report, we describe the development of an economical multiplex PCR cpe genotyping assay that works with culture lysates to distinguish among type A isolates carrying a chromosomal cpe gene, a plasmid cpe gene with a downstream IS1470-like sequence, or a plasmid cpe gene with a downstream IS1151 sequence. When this multiplex PCR assay was applied in molecular epidemiologic studies, it was found that (i) all 57 examined type A isolates with a plasmid cpe gene have either IS1470-like or IS1151 sequences downstream of the plasmid cpe gene; (ii) an IS1470-like sequence, rather than an IS1151 sequence, is more commonly present downstream of the plasmid cpe gene (particularly in North American non-food-borne human GI disease isolates); and (iii) as previously shown in the United States and Europe, isolates carrying the chromosomal cpe gene also appear to be the major cause of C. perfringens food poisoning in Japan. The superiority of this new multiplex PCR assay over existing cpe genotyping approaches should facilitate further molecular epidemiologic investigations of C. perfringens enterotoxin-associated GI illnesses and their associated cpe-positive type A isolates.