The variation at the internal transcribed spacer (ITS) region of the ribosomal DNA has been correlated with the number of nucleolar organizer regions (NORs) in some plant species. Besides, the number of NORs might influence the rate of homogenization of the rDNA repeats. In recent studies, ITS length variants were detected in bread wheat cultivars but no reports about their presence in durum wheat were found. In the present study, we localized and identified the NORs of 51 old Portuguese durum wheat cultivars by using sequential silver staining and fluorescence in situ hybridization performed with the pTa71 rDNA probe. We also detected ITS length variants by PCR-RFLP. No variation at the number of Ag-NORs per metaphase was found among the 51 durum wheat cultivars, but the PCR-RFLP technique carried out with the restriction enzyme HpaII, allowed the detection of ITS length variants among them. The molecular data was used in order to establish the genetic relationships among cultivars and botanical varieties of durum wheat. The knowledge of this feature could be useful for future design of breeding strategies, involving this collection that constitutes an excellent repository of germplasm in Portugal.