We have developed a multiplex PCR to detect bacterial disease agents of Flavobacterium psychrophilum, Vibrio anguillarum, and Pseudomonas plecoglossicida. Three primer pairs designed based on determined nucleotide sequences of the gyrB regions of these three bacteria were used. The detection limits and stringency of this method were higher enough for specific detection of these three fish diseases. Using this multiplex PCR, the rapid and simultaneous diagnosis of three major bacterial fish diseases caused by these bacteria from the organs of diseased fishes was successful.