Background: Historically, non-native species of Gambusia (Poeciliidae) have been used to control larval stages of the Asian tiger mosquito, Stegomyia albopicta Reinert, Harbach et Kitching, 2004 throughout Italy. The potential utility of indigenous populations of Aphanius fasciatus (Valenciennes) (Teleostei: Cyprinodontidae) as an appropriate alternative biological control is currently being explored. A sub-sample of ten fish collected from Cervia Saline, Italy (salinity 65 ppt; 30 degrees C) to assess their reproductive capability in captivity, harboured a moderate infection of Gyrodactylus von Nordmann, 1832 (Platyhelminthes, Monogenea). A subsequent morphological and molecular study identified this as being a new species. Results: Gyrodactylus salinae n. sp. is described from the skin, fins and gills of A. fasciatus. Light and scanning electron microscopical (SEM) examination of the opisthaptoral armature and their comparison with all other recorded species suggested morphological similarities to Gyrodactylus rugiensoides Huyse et Volckaert, 2002 from Pomatoschistus minutus (Pallas). Features of the ventral bar, however, permit its discrimination from G. rugiensoides. Sequencing of the nuclear ribosomal DNA internal transcribed spacers 1 and 2 and the 5.8S rRNA gene and a comparison with all species listed in GenBank confirmed they are unique and represent a new species (most similar to Gyrodactylus anguillae Ergens, 1960, 8.3% pair-wise distance based on 5.8S+ITS2). This represents the first species of Gyrodactylus to be described from Aphanius and, to date, has the longest ITS1 (774 bp) sequenced from any Gyrodactylus. Additional sampling of Cervia Saline throughout the year, found G. salinae n. sp. to persist in conditions ranging from 35 ppt and 5 degrees C in December to 65 ppt and 30 degrees C in July, while in captivity a low level of infection was present, even in freshwater conditions (0 ppt). Conclusions: The ability of G. salinae n. sp. to tolerate a wide range of salinities and temperatures shows its potential to readily adapt to several environmental conditions. These findings, together with the fact that A. fasciatus is a protected species and is considered as a biological control organism, necessitate further studies on the ecology and virulence of G. salinae n. sp.