One of the most prevalent pathogens found in zebrafish (Danio rerio) research facilities is the microsporidian parasite Pseudoloma neurophilia. Infections occur primarily in the spinal cord and are associated with emaciation and scoliotic changes. It is unclear why P. neurophilia is so widespread among research colonies, although transfer of infected animals and eggs between laboratories is a likely contributor. In addition to preventing the spread of this pathogen among facilities, it is desirable to have parasite-free fish for use in experiments. Therefore we have developed a polymerase chain reaction (PCR)-based diagnostic test for P. neurophilia. Compared with conventional diagnostic methods, PCR diagnosis is rapid, allows for screening of large numbers of fish, and can be applied to eggs, water filtrates, biofilms, and other samples. Using PCR primers specific to the small subunit ribosomal DNA of P. neurophilia, the test was consistently capable of detecting 10 spores per reaction and often as few as 0.1 spore per reaction, and it did not cross-react with other selected microsporidian species from fish. We recommend this PCR diagnostic assay for use by the research community to determine the presence (or absence) of P. neurophilia in colonies and for screening fish shipped between facilities, especially when parasite-free fish are required for experiments. Furthermore, we currently are using this PCR method to investigate the potential role of vertical transmission in the spread of P. neurophilia.