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Nuclear ribosomal DNA sequences encoding the 5.8S RNA and the flanking internal transcribed spacers (ITS1 and ITS2) were used to test the phylogenetic relationships within 12 Italian Quercus taxa (Fagaceae). Hypotheses of sequence orthology are tested by detailed inspection of some basic features of oak ITS sequences (i.e., general patterns of conserved domains, thermodynamic stability and predicted conformation of the secondary structure of transcripts) that also allowed more accurate sequence alignment. Analysis of ITS variation supported three monophyletic groups, corresponding to subg. Cerris, Schlerophyllodrys (=Ilex group sensu Nixon) and Quercus, as proposed by Schwarz [Feddes Rep., Sonderbeih. D, 1-200]. A derivation of the “Cerris group” from the “Ilex group” is suggested, with Q. cerris sister to the rest of the “Cerris group.” Quercus pubescens was found to be sister to the rest of the “Quercus group.” The status of hybrispecies of Q. crenata (Q. cerris × Q. suber) and Q. morisii (Q. ilex × Q. suber) was evaluated and discussed. Finally, the phylogenetic position of the Italian species in a broader context of the genus is presented. The utility of the ITS marker to assess the molecular systematics of oaks is therefore confirmed. The importance of Italy as a region with a high degree of diversity at the population and genetic level is discussed.