Monilinia spp. are responsible for brown rot decay of stone and pome fruit in the field as well as in postharvest. Monilinia laxa and M. fructigena are considered indigenous to Europe, while M. fructicola is a quarantine pathogen in the European and Mediterranean Plant Protection Organization area included in the A2 List. In Italy, it was first reported in 2009 in Piedmont (northern Italy) and rapidly spread to central Italy. We carried out a monitoring program on the occurrence of Monilinia spp. in southern Italy and a comparative characterization of the three main fungal pathogens. Molecular assays based on direct polymerase chain reaction (PCR) and real-time quantitative PCR for molecular identification of Monilinia spp. from rotted fruit were set up, validated, and applied in a monitoring program. Of the tested 519 isolates from 26 orchards, 388 (74.8%) were identified as M. fructicola, 118 (22.7%) as M. laxa, 10 (1.9%) as M. fructigena, and 3 (0.6%) were M. polystroma. M. fructicola colonies grew faster and had a higher optimal temperature for growth (26°C) than M. laxa (23°C) and M. fructigena (20°C). No relevant difference in virulence could be observed on artificially inoculated apricot, cherry, and peach fruit. The fungal species showed different responses to fungicides, because M. fructicola was more sensitive than M. laxa, especially to cyflufenamid, and M. fructigena revealed a lower sensitivity to succinate dehydrogenase inhibitors (boscalid, fluopyram, and fluxapyroxad) and quinone outside inhibitors (mandestrobin). In summary, the two species M. fructicola and M. polystroma were first detected in southern Italy where M. fructicola has largely displaced the two indigenous pathogens M. laxa and M. fructigena; the relative proportions of the three pathogens in orchards should be considered when defining the management of brown rot of stone fruit due to differences in their responses to fungicides.