Fasioliasis is one of the familiar veterinary and human health problems, with reported cases in Egypt. A simple and rapid polymerase chain reaction / restriction fragment length polymorphism (PCR/RFLPs) assay, using the common restriction endonucleases Aval, EcoRl, Eael, Sacll and Avall, is described to differentiate between both Fasciolid species. The five restriction endonucleases have been used to differentiate between the two species and based on – 1950 bp long sequence of the (18S) nuclear small subunit ribosomal RNA gene. Aval and EcoRI restriction endonucleases did not differentiate between the Fasciola isolates when each restriction enzyme gave the same restriction patterns with both species. F. hepatica and F. gigantica were well-differentiated when their small subunit ribosomal DNA were digested with Eael and Sacll restriction endonucleases. Avall restriction enzyme could be used as a genetic marker for F. hepatica when the enzyme uniquely fragmented the SrRNA gene into two bands without digesting the gene of F. gigantica.