Abstract: To provide basic molecular cytogenetic data for Sagittaria trifolia L., single- and dual-color fluorescence in situ hybridization (FISH) analysis on metaphase chromosomes of S.trifolia with 45S rDNA and Arabidopsis-type telomere sequences were performed, the expression of the 45S rDNA sites was detected using silver staining, and the karyotype of this species was established by combining chromosome measurements and rDNA FISH signals. The mean haploid karyotype length was 76.90±1.38 μm, the longest chromosome pair was 11.55±0.10 μm and the shortest chromosome pair was 4.54±0.27 μm. The karyotype asymmetry indices, CI, A1, A2, As K(%) and AI were 19.86±11.06, 0.72, 0.27, 78.82 and 15.29, respectively, and the asymmetric karyotype belonged to the 3B type of Stebbins' category. Three pairs of 45S rDNA sites were identified and located at the terminals of the short arms of chromosome pairs 8, 9 and 10, respectively. FISH signals from the Arabidopsis-type telomere sequence were observed at the ends of the short and long arms of each chromosome pair. Silver staining showed six Ag-NORs on mitotic chromosomes and one to six nucleoli in interphase cells, indicating that the three pairs of 45S sites were all expressed at interphase.