The intermediate hosts of S. mansoni in South America, B. glabrata, B. tenagophila, and B. straminea, were identified by restriction fragment length polymorphism (RFLP) analysis of the internal transcribed spacer region of the rRNA gene. The restriction patterns obtained with DdeI were the most informative of the eight enzymes that were tried. The RFLP profiles obtained using this enzyme are highly distinctive and exhibit low levels of intraspecific polymorphism even between specimens collected from diverse regions of Brazil, Argentine, Paraguay, and Uruguay. The method proved useful for the identification of DNA extracted from eggs, permitting species identification while preserving the living adult specimens for further studies.