Adhesion gene （ahal）, aerolysin gene （aerA） and cytotonic enterotoxin gene （alt） were considered as major virulence genes of Aeromonas. According to ahal, aerA and alt gene sequences, a multiplex polymerase chain reaction （MPCR） was developed to detect the above virulence genes simultaneously. The ahal, aerA and adt genes were 1087 bp, 721 bp and 480 bp PCR products respectively. Digested with EcoRV, BamnHI and Fbal, the sizes of digestive fragments of each PCR product were consistent with expectation. Using the same pairs, no PCR product was detected from S. aureus, P. putide, V. minicus and unpathogenic Aeromonads. The sensitivity of the MPCR was 100 CFU?mL^-1 of the bacteria. At the same time, multiplex PCR analysis was performed on 15 strains Aeromonas spp. isolated from different aquatic animals in Anhui and the results showed there were 10 strains with virulence genotype alt^＋ ahal^＋ aerA^＋ , 2 strains with virulence genotype alt^＋ ahal^- aerA^- and 1 strain with virulence genotype alt^＋ ahal^＋ aerA^- within 13 strains pathogenic Aeromonas spp. The positive rates of alt, ahal and aerA genes were 100%, 84. 62% and 76.92% respectively. These results indicate that the major virulence genotype of Anhui isolation strains is the high virulence phenotype of alt^＋ ahal^＋ aerA^＋ and the virulence gene alt generally exists in different Aeromonas phenospecies.