We developed and validated conventional and quantitative real-time PCR assays for the detection of DNA from the myxozoan parasite Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in fish. Assays were tested on fish tissue and on field-collected water samples to assess diagnostic and environmental DNA capabilities. The specificity, sensitivity, and broad applicability of the present assays surpass previous methods for detecting T. bryosalmonae DNA from fish tissue and water samples.