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Sex chromosomes are relatively rare in plants and morphologically distinct sex chromosomes were characterized in only a dozen species in five genera: Cannabis, Humulus, Rumex, Silene and Coccinia. The origin of sex chromosome dimorphism in plants seems to be recent, and it is believed that sex chromosomes arose in plants many times independently. Characterization of new species with distinct sex chromosomes provides an opportunity for the study of the origin of sex chromosomes and the pattern of their evolution in plants. Dioecious sea-buckthorn (2n=22+XX – female; 2n=22+XY – male) was karyotyped using chromosome measurements, fluorescence in situ hybridization (FISH) with pTa71 (45S rDNA), pCT4.2 (5S rDNA), telomeric repeats probes, C-banding/DAPI, and genomic in situ hybridization (GISH). The GISH with total genomic DNA as a probe revealed clear visualization of the Y chromosome. The strong GISH signal painted heterochromatic arm of the middle sized Y chromosome. The X chromosome was identified as one of the biggest chromosomes in the karyotype. The location of pseudoautosomal region (PAR) was established. To our knowledge the sea-buckthorn, addition to Humulus lupulus, is a second species with the much smaller Y chromosome compared to the X chromosome.