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Aeromonas hydrophila is a ubiquitous important bacterial pathogen, which is Gram-negative, motile and rod shaped. It has been associated with several diseases in fish. A. hydrophila secretes many virulence factors. These virulence can be used for detection and characterization of the bacteria. Hemolysin is a significant virulence factor of A. hydrophila. Isolation of A. hydrophila from aquatic environment has been reported by biochemical tests (Rathore et al., 2005). Previously, the detection of A. hydrophila by amplification of hemolysin gene through PCR has been done (Xia et al., 2004) and lipase H3 gene (Swaminathan et al., 2004). The present report describes the development of new species - specific PCR primers for amplification of the hole forming hemolysin gene of A. hydrophila isolates.