Microsporidian parasites are easily detected by light microscopy when infections are heavy and spores are present. However, early infections without spores, or light infections with low numbers of spores, are easily missed. This limitation has made it difficult to conduct investigations into microsporidian prevalence and transmission. In this study, we developed a quantitative TaqMan polymerase chain reaction assay to assess the presence of Ovipleistophora ovariae in the tissues of the cyprinid fish Notemigonus crysoleucas (golden shiner). The efficiency of the primer set was 100.8%, with a correlation coefficient of threshold position to copy number of 0.997 over 9 logs using a plasmid containing the cloned reaction product. No product was produced from other closely related microsporidian species (Nucleospora salmonis, Pseudoloma neurophila, Glugea stephani, Heterosporis sp., and O. mirandella). The coefficient of variation for replicate assays done on different days was 12.4%. The assay detects O. ovariae reliably at less than 10 genomic copies and 0.14 spores per reaction, but maximum sensitivity is only achieved when sonication is included as part of the DNA purification step. Using the assay, we found 4.44 × 101 to 7.91 × 106 copies μg-1 host DNA in female golden shiners, with the spore density increasing during the spawning season. The parasite was also detected for the first time in the testes of male golden shiners at 2.60 × 101 to 8.62 × 10 2 copies μg-1 host DNA.