The fluorescent amplified fragment length polymorphism method (AFLP) has been successfully applied to one of the brown rot fungi species — Monilinia fructicola, which causes severe losses in stone fruit production. This is the first report on the use of AFLP methodology for studying genetic variability among different M. fructicola isolates. A total of 1256 scorable, well defined AFLP fragments were obtained with 20 primer pairs, of which 462 were polymorphic. An unweighted pair group (UPGMA) dendrogram was constructed based on Jaccard’s similarity coefficient. A high similarity of the two Malus isolates from the USA was observed, while the other three Prunus isolates, from Spain, Japan and New Zealand, formed a second, more variable group. Comparison of AFLP fingerprinting data between M. fructicola and M. laxa revealed completely different banding patterns and demonstrated the suitability of the AFLP marker system for cross species differentiation of brown rot fungi.