The internal transcribed spacers (ITS) between the 18S and the 28S rRNA genes of 4 M. laxa and M. fructigena isolates from France was amplified by polymerase chain reaction (PCR), and sequenced. Multiple alignment of ITS sequences, and comparison with published sequences showed very little intraspecific variability. However, low interspecific polymorphism was observed in 2 regions, ITS1 and ITS2, respectively. Species-specific PCR primers were designed to amplify a 356-bp fragment for each of the three species. The specificity of each primer pair was assessed successfully in a collection of 17 M. laxa isolates, 18 M. fructigena isolates and 6 M. fructicola isolates from different hosts. Using stringent PCR conditions, no cross-reaction was observed with tested isolates. Test specificity was verified with total DNA from different fungal species which were either phylogenetically related to Monilia or frequently isolated from affected fruits. Using this new reliable technique, doubtful isolates can be directly identified in a single PCR run, testing them simultaneously with each of the three primer pairs. Moreover, the Monilinia sp. were successfully detected and identified directly on symptomatic fruits. This simple and rapid method could be particularly useful to detect M. fructicola, a listed quarantine pest for Europe.