Fungi of the Monilinia genus occur worldwide and affect a wide range of economically important stone fruits. Several Monilinia species are responsible for brown rot. Although this disease is common in Brazil, Monilinia sp. genetic variability in Brazilian orchards has generally been poorly characterized. The present study represents the first report on the genetic diversity of Monilinia sp. from Brazilian orchards. The genetic structure of the Brazilian population was also compared to isolates from other countries, together with some morphological characteristics and aggressiveness. Sixty-one isolates belonging to the Monilinia genus were obtained from different orchards in Brazilian states. Ten Monilinia fructicola isolates from the United States and one isolate from a fruit imported into Brazil were also evaluated. Phylogenetic analysis of the ITS1-5.8S-ITS2 region (internal transcribed spacer) clustered most Brazilian and American isolates with M. fructicola authentic strains from Q-Bank. Two isolates (one from an imported fruit) clustered as Monilinia laxa. The results revealed M. fructicola as the prevalent species associated with brown rot in Brazilian orchards. To evaluate the intraspecific diversity of M. fructicola and M. laxa, multigene sequence analysis was performed using ITS1-5.8S-ITS2 and TEF1 (elongation factor 1). Whilst TEF1 is the most phylogenetically informative gene for intraspecific studies of M. fructicola, RPB2 (RNA polymerase II gene) displayed low variation in intraspecific analysis, but was an informative locus for assigning isolates to M. fructicola or M. laxa species. The AMOVA suggests that Brazilian isolates from the States of the main producing regions belong to a single genetic population, which is genetically distinct from the US (Californian) population of M. fructicola.