In order to elucidate the interspecific relationships of the genus Pulsatilla, a molecular cytogenetic study was carried out on the five species (P. cernua, P. davurica, P. koreana, P. tongkangensis, and P. chinensis) of the genus Pulsatilla distributed throught the Korean Peninsula. The Somatic methaphase chromosome numbers of all five species were 2n = 2x = 16. The 5S and 45S rDNA genes were ... [Show full abstract] physically mapped on the metaphase chromosomes using fluorescence in situ hybridization (FISH). The 5S rDNA genes were detected on the long arm of chromosome 4 and on the long arm of chromosome 7, In addition, the 45S rDNA genes were localized on the short arms of chromosomes 7 and 8 in P. cernua, P. davurica and P. chinensis. An additional locus of the 5S rDNA gene was detected on the chromosome 6 in P. koreana. One of the 5S rDNA loci was not found on the homologue of chromosome 4 in P. tongkangenesis. A polymorphism of the signal intensity was also detected on the chromosomes 7 and 8 in P. tongkangenesis. Sequence analysis showed that the 5S rDNA sequences varied between species, ranging from 495 to 535 bp. The sequence data indicated that P. koreana was distantly related to the other four species of Pulsatilla.