Macrobrachium rosenbergiinodavirus (MrNV) and Macrobrachium rosenbergii dicistrovirus (MrDV) are the main viral pathogens of Macrobrachium rosenbergii. In order to detect the two viruses, two pairs of primers targeted sequences located within the MrNV capsid protein gene and MrDV RNA dependent RNA polymerase (RDRP) gene amplified amplicons of 384 bp and 472 bp, respectively. The reaction conditions of the duplex RT-PCR were optimized and PCR products were sequenced. Meanwhile, the specificity and sensitivity of duplex RT PCR were studied. Results reveal that optimum annealing temperature of duplex RT PCR is 60 ℃ and the minimum concentrations of primers are 0.1 μmol/L of MrNV384 and 0.05μmol/L of MrDV472. The detection limit of duplex RT PCR was determined to be 360 fg of tissue total RNA for both the viruses. The two pairs of primers were found to be specific to MrNV and MrDV respectively, and did not to react to either Taura syndrome virus (TSV), White spot syndrome virus (WSSV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), or Aeromonas hydrophila TPS-30. Sequence analysis of RDRP gene amplicons of MrDV revealed no variations. By contrast, sequence analysis of MrNV amplicons showed many variations in nucleotide sequences of the capsid protein gene. The phylogenetic tree shows that the stains of MrNV, collected from Yangtze River delta in 2011, are from two genotypes, Chinese and Southeast Asia genotypes. This new duplex RT PCR method could be applied for routine health monitoring, early virus detection, and studying virus host interaction, detection of carriers and screening of broodstock.