The object of this work was to analyze the karyotype structure of <i>Rumex thyrsiflorus</i> using differential fluorescent methods of chromosome staining (C-banding/DAPI and CMA₃/DA/DAPI) and molecular sex markers. The results obtained were compared with data on the structure of the sex chromosomes and autosomes in <i>R. acetosa,</i> a model species in studies of sex determination and sex chromosome evolution in plants with an XX/XY₁Y₂ system. A high level of similarity was found in the sex chromosome structure of the 2 species, along with small differences in their autosomal complexes. It suggests that differentiation of these 2 closely related species was not accompanied by major structural changes within their sex chromosomes. Molecular tests, however, revealed differences in the composition of male-specific repetitive sequence RAYSII, occurring in the Y₁ chromosome. Amplification of this sequence showed the presence of a single product (∼700 bp) in <i>R. acetosa</i> and of 2 products (∼600 bp and ∼700 bp) in <i>R. thyrsiflorus</i>. The longer product (∼700 bp) was also revealed in <i>R. arifolius</i>, another species closely related to <i>R. acetosa</i>. The shorter DNA fragment, characteristic of <i>R. thyrsiflorus</i>, differed from the common product by of a large indel with a length of 110 bp. This fragment may serve as a species-specific molecular marker useful in taxonomical and population studies as well as in further research on the sex chromosome differentiation in <i>R. thyrsiflorus</i>.