Chrysanthemum is one of the most popular and economically important ornamental plants due to its huge diversity in growing habits, wide range of colors, and different patterns of flower. In the present study, we conducted the karyotype analysis in four naturally occurring genotypes of Chrysanthemum boreale. Karyotype studies based on the fluorescence in situ hybridization (FISH) using 5S and 45S rDNAs. Four chrysanthemum genoteyps showed an aneuploid chromosome number of 2n=18+2 (111016 and 111021) or a diploid of 2n=18 (121001 and 121002). All the genoteyps had the same karyotype formula of 14 metacentrics and 4 submetacentrics. In 111016, the chromosome length during somatic metaphase ranged from 3.11 ± 0.26 μm (shortest) to 3.94 ± 0.20 μm (longest), with a total length of 32.94 μm. The chromosome length at the mitotic metaphase ranged from 3.11 to 6.46 μm, with a total length of 32.94 μm in 111016 and 51.05, 32.81, and 46.00 μm in 111021, 121001, and 121002, respectively. The 5S rDNA and 45S rDNA signals recorded different in all four wildly grown genotypes of C. boreale. This information can be useful in cultivar improvement, as well as in elucidation of the evolution of the chrysanthemum.