Fasciolosis, caused by the trematode Fasciola hepatica, is a zoonosis of economic importance in livestock that is emerging as a chronic disease in humans. The intermediate hosts are lymnaeid snails, in which diagnosis of infection is traditionally based on cercarial shedding, tissue sectioning and crushing. We developed a PCR assay for the sensitive and specific detection of F. hepatica in field-collected Lymnaea sp. snails. A primer pair was designed to amplify a 405 bp fragment of the cytochrome c oxidase subunit 1 gene of F. hepatica. The PCR assay showed a limit of detection of 10 pg of genomic F. hepatica DNA. No cross-reactions were observed with samples from other related trematode species or from the snail hosts Lymnaea columella and Lymnaea viatrix. DNA sequencing of the amplicon showed 100% homology with F. hepatica, and 75-89% homology with other trematodes on regions that did not include the entire set of primers. Two samples from Argentina were analysed. For snails in sample 1 (n = 240), identified as L. columella, the infection rate was 17.5 and 51.3% by direct examination and PCR, respectively. For snails in sample 2 (n = 34), identified as L. viatrix, the infection rate was 2.9 and 61.8% by direct examination and PCR, respectively. Differences in infection rates between these diagnosis methods were significant for both samples. Our PCR technique showed to be effective for detecting specific F. hepatica infections of low intensity in the intermediate host, and hence it could be used to study the epidemiological situation in a given area, as well as to assess host suitability for the parasite.