To detect marine birnaviruses (MBV) from fish samples, a two-step PCR assay was developed. The first step is a reverse transcription (RT) PCR using a primer set design based on the infectious pancreatic necrosis virus (IPNV) gene. The second step is a nested PCR using a primer set design based on the VP2/NS junction region of MBV. This method was specific for aquatic birnaviruses; heightened sensitivity was indicated in that 1 fg of viral genome could be detected when nested PCR was used. The birnavirus gene was detected from clinical samples with a high ratio even though the samples gave negative results in virus isolation. This method could be useful to survey MBV not only in diseased fish but also in nonsymptomatic carriers or reservoirs.