The aim of this work was to cytogenetically characterize triticale cultivars through fluorescence in situ hybridization (FISH) analysis of their rye chromosomes. In the present work, we studied six cultivars of triticale ('Cayú-UNRC', 'Cumé-UNRC', 'Genú-UNRC', 'Ñinca-UNRC', 'Quiñé-UNRC', and 'Tizné-UNRC'), released by the Universidad Nacional de Río Cuarto (UNRC), Córdoba, Argentina. The cultivars were obtained from the International Center for the Improvement of Maize and Wheat (CIMMYT) and improved for fresh forage, haymaking, and feed grain at UNRC. The distribution and organization of highly repetitive DNA sequences of Secale cereale (pSc74, pSc200, pSc250, and pSc119.2) using FISH analyses revealed a specific localization of the signals for several rye chromosomes, which allowed us to distinguish the cultivars. Cluster analysis showed a great cytogenetic similarity among the rye cultivars used to originate these hybrids. The knowledge of the variability among triticale cultivars is necessary to propose future crosses in breeding programs. This study will also be valuable to identify commercial seeds and to analyze the possible association between agronomic characters and the presence of certain rye chromosomes or specific regions in these chromosomes.