To prevent the entry and spread of the brown rot fungus Monilinia fructicola in Europe, a fast and reliable method for detection of this organism is essential. In this study, an automated DNA extraction method combined with a multiplex real-time PCR based on TaqMan chemistry was developed for fast, convenient and reliable detection of both the EU quarantine organism Monilinia fructicola and the three other brown rot fungi M. fructigena, M. laxa and Monilia polystroma. Using the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene repeat, a Monilinia genus-specific primer pair and two differently labelled fluorogenic probes specific for M. fructicola and the group M. fructigena ⁄ M. laxa ⁄ Monilia polystroma were developed. The analytical specificity of the assay was assessed by testing 33 isolates of the four brown rot fungi and 13 isolates of related fungal species or other fungal species that can be present on stone and pome fruit. No crossreactions were observed. The assay was found to have a detection limit of 0AE6 pg of DNA, corresponding to 27 haploid genomes or four conidia. Comparison of a manual DNA isolation followed by a conventional PCR with an automated DNA isolation combined with the presently developed real-time PCR showed that the latter method gave improved results when tested with 72 naturally infected stone fruit samples. The detection rate increased from 65 to 97%.