A one-step reverse transcriptase–polymerase chain reaction (RT-PCR) method was developed and optimized for the detection of duck hepatitis virus type 1 (DHV-1) using the Viral Gene-spin™ viral DNA/RNA extraction kit. A pair of DHV-1–specific primers was designed against the gene encoding RNA-dependent RNA polymerase (3D gene). Using RNA prepared from duckling liver samples infected with two reference and seven Korean field isolates of DHV-1, one-step RT-PCR with DHV-1–specific primers amplified a 467-bp fragment. Under the same conditions, no amplification was observed for 14 other avian pathogenic viruses and bacteria. Using RNA prepared from serial dilutions of the DHV-1 with the supernatant of the uninfected duckling liver homogenate (10% w/v), the one-step RT-PCR assay was found to be sensitive to 10 50% egg lethal dose (ELD₅₀) 0.1 ml⁻¹ of DHV-1. Furthermore, this method detected DHV-1 from the livers and allantoic fluid of duck embryos dying before 3 days postinoculation (PI) and of chicken embryos that were chilled at 3 days PI. Therefore, this one-step RT-PCR method is rapid, sensitive, and reliable, and can be readily adapted for detection of DHV-1 from other clinical samples.