Environmental DNA (eDNA) surveys are increasingly used to inform management decisions for non-native species, for example, by detecting the presence and plotting distributions of species that may be in too low abundance for easy detection by conventional means. A recently-developed nested PCR protocol was used to assess the distributions of three non-native fish species in two river basins of southern England (River Test, Hampshire; River Ouse, Sussex). These river basins were known to contain three non-native fishes, either in the recent past or currently: two invasive small-bodied fish species (topmouth gudgeon Pseudorasbora parva, sunbleak Leucaspius delineatus), as well as a currently non-invasive species predicted to become invasive under future climate conditions, pumpkinseed Lepomis gibbosus. Water samples were collected at locations from headwater streams to estuary. Pumpkinseed and sunbleak were both detected downstream of an angling venue in the Sussex Ouse catchment known to contain those species, with an upstream expansion of sunbleak suggested by the detection of eDNA at a few upstream locations. Neither sunbleak nor topmouth gudgeon was detected in water samples from the River Test catchment, suggesting that neither species has persistent populations in that river catchment.