An RT-PCR detection technique was developed for Pholobacterium damsela (=Photobacterium damsela subsp. damsela and Ph. damsela subsp. piscicida) from apparently healthy yellowtail (Seriola quinqueradiata) and other wild fishes, based on the analysis of a species-specific sequence in 16S rRNA of Ph. damsela. Specificity of the RT-PCR with K-F1 (sense primer) and R-GEN (antisense primer) was confirmed by using Ph. damsela subsp. damsela and Ph. damsela subsp. piscicida, 12 strains of fish—associated Vibrio species. Sensitivity of the RT-PCR was improved by combining a pre-culture of the sample (spleen homogenate) with tryptic soy broth (NaCl 2%). This RT-PCR revealed the presence of Ph. damsela from wild yellowtail, filefish (Thamnaconus modestus) and horse mackerel (Trachurus japonicas) caught in the sea as well as apparently healthy cultured yellowtail.