The duck industry is at high risk from duck viral hepatitis (DVH) and has the potential to cause substantial financial losses because of the high mortality rates observed in duck farming, even with continuous breeder duck flock vaccination. Among the different etiological agents of DVH, duck hepatitis A virus type-1 (DHAV-1) is the most common followed by DHAV-2 and DHAV-3. Although DHAV-I is more common and pathogenic, DHAV-3 has just emerged from duck farms in North Egypt, thus there's a pressing need to find a way to detect both DHAV-1 and DHAV-3 rapidly and simultaneously using real-time qPCR. To assess and compare the sensitivity of the real time reverse transcriptase PCR (rRT-PCR) technique for the detection of DHAV-3 and DHAV-1, dilution range of titrated DHAV-1 and DHAV-3 reference strains from 10^7.2 and 10⁶ EID₅₀/ml to 1EID_50, was implemented, respectively. The results of the current study confirmed that the rRT-PCR assay's had lowest detection limit for DHAV-1 and DHAV-3 was 10^2.2 and 10² EID₅₀/ml, respectively, and it is ten-fold higher than RT-PCR. The rRT-PCR was highly specific to DHAV-1 and DHAV-3, as other avian diseases and nucleic acid isolated from samples that tested negative for DHAV. When examining clinical samples for rRT-PCR, the diagnostic sensitivity was better than the RT-PCR. It detected 25 out of 40 clinical suspected samples but the RT-PCR detected only 15 out of 40 clinical suspected samples. In conclusion, the assay may be used as an efficient, rapid, sensitive, specific, and focused molecular diagnostic technique for detection and epidemiological investigations of DVH caused by both DHAV-1 and DHAV-3.