AbstractAnguillid herpesvirus 1 (AngHV1) causes a haemorrhagic disease with increased mortality in wild and farmed European eel, Anguilla anguilla (L.) and Japanese eel Anguilla japonica, Temminck & Schlegel). Detection of AngHV1 is currently based on virus isolation in cell culture, antibody‐based typing assays or conventional PCR. We developed, optimized and concisely validated a diagnostic TaqMan probe based real‐time PCR assay for the detection of AngHV1. The primers and probe target AngHV1 open reading frame 57, encoding the capsid protease and scaffold protein. Compared to conventional PCR, the developed real‐time PCR is faster, less labour‐intensive and has a reduced risk of cross‐contamination. The real‐time PCR assay was shown to be analytically sensitive and specific and has a high repeatability, efficiency and r2‐value. The diagnostic performance of the assay was determined by testing 10% w/v organ suspensions and virus cultures from wild and farmed European eels from the Netherlands by conventional and real‐time PCR. The developed real‐time PCR assay is a useful tool for the rapid and sensitive detection of AngHV1 in 10% w/v organ suspensions from wild and farmed European eels.