ABSTRACT The internal transcribed spacers (ITS1) of nuclear ribosomal DNA of ten populations of Italian subspecies of Paeonia mascula s.l. were analysed. The aim was to correlate molecular and morphological data in order to solve taxonomic and distributive problems. Three highly homogeneous patterns of molecular variability and three corresponding groups of populations were identified. One group includes plants with both molecular and morphological similarities suggesting their attribution to P. mascula ssp. russoi. The other two groups show differences which do not grant their present separation in the two subspecies P. mascula ssp. mascula and P. mascula ssp. hellenica. This study confirms that reticulate evolution obscured morphological variability in Paeonia and stresses the usefulness of ITS analysis in studies at low taxonomic circumscriptions.