Acknowledgements to Dr Kentaro Yoshimura, Department of Parasitology, Medical School of University of Akita, Japan, for providing us with Angiostrongylus cantonensis; Joziana Barçante, Department of Parasitology of Federal University of Minas Gerais, Brazil, for maintaining the life cycle of A. vasorum; Ester Maria Mota, Department of Pathology, Oswaldo Cruz Institute-Fiocruz, Rio de Janeiro, Brazil, for being in charge of A. costaricensis life cycle.Angiostrongylus cantonensis, A. costaricensis, and A. vasorum are etiologic agents of human parasitic diseases. Their identification, at present, is only possible by examining the adult worm after a 40-day period following infection of vertebrate hosts with the third-stage larvae. In order to obtain a diagnostic tool to differentiate larvae and adult worm from the three referred species, polymerase chain reaction-restriction fragment length polymorphism was carried out. The rDNA second internal transcribed spacer (ITS2) and mtDNA cytochrome oxidase I regions were amplified, followed by digestion of fragments with the restriction enzymes RsaI, HapII, AluI, HaeIII, DdeI and ClaI. The enzymes RsaI and ClaI exhibited the most discriminating profiles for the differentiation of the regions COI of mtDNA and ITS2 of rDNA respectively. The methodology using such regions proved to be efficient for the specific differentiation of the three species of Angiostrongylus under study.