Interspecific F₁ hybrid (2n=30-36) was produced for the first time by artificial cross pollination between Chrysanthemum boreale (2n=18, diploid) and C. vestitum (2n=54, hexaploid). The analysis of fluorescence in situ hybridization (FISH) in the 45S and 5S rDNA sites showed that the F₁ hybrid had six and eight signals respectively (instead of eight and six signals of the 45S and 5S rDNA sites respectively) were shown in the F₁ hybrid perhaps due to translocation and recombination. Genomic in situ hybridization (GISH) distinguished respective parental chromosomes in the F₁ hybrid chromosome complement and showed that at the time of using the biotin labeled probe of C. boreale showed approximately 9-yellow-green-colored chromosomes were hybridized with the probe of C. boreale, might related to C. boreale, 18 orange-colored chromosomes might have a commone chromosome homology between C. boreale and C. vestitum, while the other 9 red-colored were not hybridized chromosomes with the probe and could be of C. vestitum.