[show abstract][hide abstract] ABSTRACT: Eleven species belonging to superfamily Ascaridoidea, which infect marine and freshwater fish, mammals, and fish-eating birds, were analyzed using a PCR-RFLP method. The following species were investigated: Anisakis pegreffi, A. physeteris, and A. simplex (parasites of fish and mammals), Contracaecum osculatum, C. radiatum, and C. rudolphi (parasites of mammals and fish-eating birds), Hysterothylacium aduncum (a parasite of fish), Porrocaecum angusticolle, P. crassum, P. depressum, and P. ensicaudatum (parasites of fish-eating birds). PCR-amplified rDNA regions encompassing ITS1, 5.8S rDNA, and ITS2 produced on templates of genomic DNA isolated from all investigated species were digested with TaqI, AluI, BsuRI, and RsaI endonucleases. Restriction patterns showed that endonuclease TaqI is the most useful enzyme for identification of all investigated species. No variations in restriction patterns within each species were detected. Therefore, we propose that the PCR-RFLP assay described in this report may be used for identification of marine and freshwater parasites from superfamily Ascaridoidea.