Technical Abstract: Nucleospora salmonis is an intranuclear microsporidian found in salmonid fishes. The parasite principally infects lymphoblast cells resulting in chronic severe lymphoblastosis and leukemia-like conditions. N. salmonis generally weakens immune system function allowing for other, opportunistic infections, but it can also be fatal. N. salmonis occurs naturally in the NW United States and poses a significant threat to commercial aquaculture species. Current methods for the detection of N. salmonis involve histochemical staining and analysis and nested PCR. In order to improve these methods we devised a real-time quantitative PCR assay and compared it existing methods in several different tissues. Our finding found the newly develop qPCR assay to be much faster and more sensitive for the detection of the pathogen in all tissues tested, with minimal detection of false positives.