Incomplete reproductive barriers between species, especially in sympatric areas where several species coexist, may result in hybridization and an increase in genetic diversity. Here we assessed the amount of genetic diversity in a community of three interfertile and sympatric European oaks (Quercus frainetto Ten., Q. petraea Liebl. Matt. and Q. pubescens Willd.) situated in central Italy. We used 11 microsatellite markers derived from Expressed Sequence Tag (EST-SSRs) and we implemented a Bayesian clustering analysis to assign individuals to species or hybrids. All genotyped loci were polymorphic for all the species and three genetic clusters corresponding to each species were detected. Significant differences and a higher level of gene flow were observed between the three oak species. Occurrence of hybrids varied markedly within the studied area: hybrids between Q. petraea and Q, pubescens were the most frequent, while hybrids between Q. petraea and Q. frainetto were particularly rare. Q. pubescens and Q. petraea showed the highest number of alleles compared to Q. frainetto, which was characterized by a low number of private, but highly frequent, alleles. However, Q. frainetto showed a lower genetic diversity and a stronger reproductive isolation from the other two oak species.