Fasciolosis is one of the neglected tropical diseases that negatively affect the farming industry and public health. The present study aimed to evaluate a polymerase chain reaction (PCR) assay for detection of infection with Fasciola sp. in field-collected Lymnaea natalensis snails from two rural localities in Egypt; and to compare between it and the ordinary microscopic methods. A total of 1000 lymnaeid snails were collected, and then the samples were examined for the infection by crushing, cercarial shedding and molecular methods. The average sensitivity of microscopic methods compared to PCR was 56.15% and this gives the indication that the molecular techniques were highly sensitive, specific and reliable methods for detecting Fasciola sp. The study detected two genes; nuclear ribosomal central internal transcribed spacer 1 (ITS1856bp) and mitochondrial cytochrome oxidase subunit I (CO1 831bp). Gel electrophoresis
showed strong bands for these single snails' samples and confirmed the presence of infection in both localities. Molecular techniques gave sensitive, accurate identification of infection, established prevalence in endemic areas and planning time for proper control strategy.