Aeromonas hydrophila is a bacterium associated with many diseases and disorders such as fin rot, skin ulcers and lethal hemorrhagic septicemia in fish. It bears several virulence factors including type III secretion system (T3SS), aerolysin, cytolytic enterotoxin and enzymes (e.g. hemolysins, lipase) that seem to play an important role in its pathogenesis. Detection of virulence markers by polymerase chain reaction (PCR) is a key procedure in defining the pathogenic ability of pathogenic bacteria and preparing a vaccine for its treatment. In this sense, this study was aimed to determine the frequency of virulence genes in isolates obtained from infected cultured carps in Khuzestan province. Out of 200 moribund carps with septicemic symptoms, 125 isolates were belonged to the motile aeromonads and 59 isolates were identified as A. hydrophila by biochemical methods. Finally, using PCR analysis, 31 isolates were identified as A. hydrophila. Five virulence genes were detected in these isolates including hemolysin, aerolysin, cytolytic enterotoxin and T3SS (aopB and ascV) by specific primers. Results showed that 23 (74.19%), 18 (58.06%), 16 (51.61%), 13 (41.63%) and 10 (32.25%) isolates possessed cytolytic enterotoxin, hemolysin, aerolysin, and T3SS genes, respectively. The results of the present study showed that among 31 isolates, only five isolates had all of dominant virulence genes. Thirteen other isolates had genotypes including hlyA+, aerA+, and act+. The remaining isolates had at least one virulence gene. This study showed that determination of the virulence genes by PCR can be a reliable method to identify a potential pathogenic Aeromonad strain.