Abstract Farming of cobia (Rachycentron canadum) is one of the important maricultured fish species in Taiwan. However, bacterial diseases have plagued cobia aquaculture industry. In this study, based on the growth characteristic, morphological, and biochemical properties of an isolated bacterium, from diseased fish of were similar to those of Photobacterium damselae subsp. piscicida. The 16S rRNA sequence showed 99% identity with P. damselae subsp. Piscicida (GenBank accession number AY147860.1). The pathogenicity experiments were tested by intraperitoneal injection (IP) in juvenile cobia (12~17 g). The LD50 value of P. damselae subsp. piscicida for cobia was 1.0¡Ñ105.6 cells/ml. In order to understand the spreading routes of P. damselae subsp. piscicida in cobia, we infected in cobia fries with P. damselae subsp. piscicida. The results showed that 6h postinfection, P. damselae subsp. piscicida was isolated from gills and gastrointestines. And at 96h P. damselae subsp. piscicida could be isolated form all organs. Histology and fluorescence in situ hybridization showed that the bacterium enters via gills or gastrointestinal tracts and spreaded to kidney and liver, and eventually developed into systemic infection. In addition, genus-specific sequence of 16S rRNA of pasteurellosis was amplified by PCR using the PDPF and PFPR primer. A 847 bp fragment was observed in all the nodules organ and bacterial suspension. The technique is capable of rapid identification of the sequence pathogen in fish.