Menyanthaceae is a family of aquatic plants in the order Asterales. There are 76 species in six genera (Liparophyllum, Nymphoides, Menyanthes, Ornduffia, Nephrophyllidium, Villarsia), some species used for medicine or edible. Menyanthes trifoliata is a monotypic genus that belongs to Menyanthaceae. This species is distributed in Northern Hemisphere, such as Asia, and Europe. It is designated as a second class rare and endangered species in Korea. Cytogenetic investigations based on chromosomes provide insight into basic genetic and genomic characteristics in species. However, genomic information based on chromosome characterization and physical mapping of cytogenetic markers has been very limited. Dual-color FISH karyotype analysis of M. trifoliata was conducted using 5S and 45S rDNA probes. The number of chromosomes is 2n = 54. The chromosome length was 1.52 to 3.37 µm, and the average chromosome length was 2.34 µm. In the FISH, M. trifoliata represents two pairs of 45S rDNA signals and one pair of 5S rDNA signals. Also, in the same condition, 5S rDNA probe was localized to chromosome #5, and 45S rDNA was located at the secondary constriction of the acrocentric satellite chromosomes #3 and #24. These results would be used as basic data for genetic diversity and the preservation and restoration of endangered species.