Species-specific polymerase chain reaction (PCR) targeting the 16S rRNA gene of Nocardia seriolae was developed.The PCR targeted nucleotide #609 to 1038 (Escherichia coli numbering), which gave a 432 bp-length product. This method could detectN. seriolaetype strain (JCM3360) and eight clinical isolates of this species from yellowtailSeriola quinqueradiataand Japanese flounder Paralichthys olivaceus, but not those of other bacterial species including 5 otherNocardiaspp. and 4 yellowtail pathogens. The detection limit of the PCR was 102CFU. Eight diseased yellowtail were employed for detection of the bacterium by the PCR. Positive results were obtained from all fish.