Rumex hastatulus is the North American endemic dioecious plant with heteromorphic sex chromosomes. It is differentiated into two chromosomal races: Texas (T) race characterised by a simple XX/XY sex chromosome system and North Carolina (NC) race with a polymorphic XX/XY1Y2 sex chromosome system. The gross karyotype morphology in NC race resembles the derived type, but chromosomal changes that occurred during its evolution are poorly understood. Our C-banding/DAPI and fluorescence in situ hybridization (FISH) experiments demonstrated that Y chromosomes of both races are enriched in DAPI-positive sequences and that the emergence of polymorphic sex chromosome system was accompanied by the break of ancestral Y chromosome and switch in the localization of 5S rDNA, from autosomes to sex chromosomes (X and Y2). Two contrasting domains were detected within North Carolina Y chromosomes: the older, highly heterochromatinised, inherited from the original Y chromosome and the younger, euchromatic, representing translocated autosomal material. The flow-cytometric DNA estimation showed ∼3.5 % genome downsizing in the North Carolina race. Our results are in contradiction to earlier reports on the lack of heterochromatin within Y chromosomes of this species and enable unambiguous identification of autosomes involved in the autosome-heterosome translocation, providing useful chromosome landmarks for further studies on the karyotype and sex chromosome differentiation in this species.