Hemolysin is an important pathogenic agent of Edwardsiella tarda (ET). A fragment containing hemolytic gene, of which sequence is 4,264 bp, was cloned from ET-12 chromosome by shutgun method. It has no similarity to the hemolysin genes reported, of which 424 bp of open read frame (ORF) 3 has 68% similarity to the hemolysin regulatory gene of S. typhi (slyA). The subclone which has complete ORF3 was hemolytic, and the other of which Kanamycin gene was inserted in Pvu I of ORF3 was no hemolytic. It proved the gene was in chromosomes of ET-12 and the other ET strains by hybridization in situ and Southern blot. As recombination plasmid with the gene entered nonhemolytic ET by electroporation, no hemolytic phenomenon was observed. Conclusion was that the gene wasn't the hemolysin gene and was hemolytic relative gene.