Aims Under intensive and stressful aquaculture conditions, cultured eels are highly susceptible to virulent Aeromonas sp. infections. To rapidly and simultaneously confirm Aeromonas isolate and its virulence, a two-tube multiplex PCR (mPCR) assay incorporating gyrB gene for genus-specific recognition and seven major virulence genes for virulence assessment was developed. Methods and Results Eight pairs of primers were designed and divided into two groups-gyrB, ahpA, epr and aerA in tube 1 and alt, act, ast and hlyA in tube 2. The optimized mPCR conditions were the same except for their final concentrations. The specificity of the mPCR was validated by the extracted DNA of 10 Aeromonas and 8 non-Aeromonas species, or mixed DNA templates. Detection limits were determined to be 200 copies per mu l in tube 1 and 20 copies per mu l in tube 2. The mPCR reproducibility was tested by both artificial challenge and clinical samples. Conclusions The results showed this two-tube mPCR assay was rapid, specific, sensitive and reliable. Significance and Impact of the Study To our knowledge, this is the first report to distinguish virulent Aeromonas isolates from nonvirulent ones by seven popular and major virulence genes at the genus-specific level. And it will be useful for large-scale screening of virulent Aeromonas sp. in cultured eels.