Kudoa septempunctata, a myxosporean parasite, was recently identified as the causative agent of food poisoning resulting from the consumption of raw olive flounder (Paralichthys olivaceus). A single blind inter-laboratory study, involving 5 laboratories, was conducted to validate a quantitative real-time PCR assay for the detection of the parasite. We obtained relatively constant values for log rDNA copies/g from these laboratory analyses (SD = 0.35-0.86), suggesting the validity of the real-time PCR method for the detection of K. septempunctata in P. olivaceus. Detection of K. septempunctata in muscle tissue samples collected from both sides of the fish indicated that K. septempunctata infection spreads throughout the body of P. olivaceus. K. septempunctata infection in P. olivaceus is thought to occur during the early stage of fish growth because a K. septempunctata gene was detected in 1 of 300 P. olivaceus fry tested. Feeds seem not to be sources of infection. To prevent food poisoning due to K. septempunctata, the mechanism of infection and proliferation of K. septempunctata in P. olivaceus should be elucidated, and other hosts of the parasite should be identified. The sensitive real-time PCR method described here will be a useful tool for resolving these issues.