The data are used to support the development and validation of two quantitative polymerase chain reaction (qPCR) assays to detect the parasite Nanophyetus salmincola DNA in water samples and fish and snail tissues. The data link to a series of experiments that are described in the publications. Experiment 1 defines the linearity and detection limits of the assays. Experiment 2 assesses the utility of the assays to detect N. salminicola waterborne cercaria in one liter volumes of water. Experiment 4 assesses the detection of N. salminicola waterborne cercaria in small volumes (50 microliters). Experiments 5 and 6 assesses the within and between run repeatability of the assays. Experiment 7 assesses the diagnostic specificity of the assays in a fish population known to be negative for the parasite. Experiment 8 assesses the diagnostic specificity of the assays in a fish population known to be positive for the parasite. Experiment 9 assesses the diagnostic accuracy of the assays in fish artificially exposed to the parasite. Experiment 10 assesses the ability of the assay to detect the parasite in the intermediate snail host (Juga spp.). Experiment 11 assesses the ability of the assay to detect the parasite in the one liter volumes of river water collected at sites where the intermediate snail host is either present or absent.