Brown rot, caused by Monilinia spp., is an economically important pre- and postharvest disease of pome and stone fruits worldwide. In Serbia, apple is the most widely grown pome fruit, and the distribution of economically important Monilinia spp. responsible for apple brown rot is unknown. Hence, we conducted a three year survey, from 2010 to 2012, where 349 isolates were obtained from six orchards and four storage facilities from five different apple cultivars with brown rot symptoms. Morphological characterization of the isolates, multiplex PCR, and phylogenetic analysis revealed four species: M. fructigena, M. laxa, M. fructicola, and Monilia polystroma. All species were found in the orchard and in storage, with M. fructigena predominating, followed by M. polystroma. Representative isolates were analyzed in vitro and in vivo where differences in growth rate, sporulation, and virulence on apple fruit were observed. Findings from this investigation demonstrate diversity in the species responsible for pre- and postharvest apple brown rot, which has significant implications for pathogen detection and for developing disease-specific management strategies.