In 30 Fagaceae individuals from the Castanea, Fagus and Quercus genera, the internal transcribed spacer region (ITS) of the ribosomal DNA (rDNA) was amplified and digested with four restriction enzymes (HaeIII, HpaII, Sau96I, and TaqI), producing ITS PCR-RFLP markers. This technique allowed the discrimination of the Fagaceae species according to genus, infrageneric group and ecological area. This study constitutes the first application of the alternative co-dominant marker system ITS PCR-RFLP in Fagaceae species and proved to be highly suitable for taxonomic and ecological discrimination, constituting a useful molecular tool for taxonomists and forestry researchers.