Abstract: Chromosomal architecture and ribosomal DNA organization were investigated in Polygonatum sibiricum Red. and P. cyrtonema Hua using conventional squash preparation combined with fluorescence in situ hybridization (FISH) targeting 45S rDNA. Results showed that: (1) P. sibiricum exhibited a karyotype formula of 2n=2x=24=2m+8sm+14st(2SAT), corresponding to type 3B. Two pairs of 45S rDNA loci were identified, located on the long arm of chromosome 3 and the short arm of chromosome 6, respectively. (2) P. cyrtonema exhibited a karyotype formula of 2n=2x=22=6m+12sm(2SAT)+4st(2SAT), corresponding to type 3B. Three pairs of 45S rDNA loci were identified, located on the long arms of chromosomes 1, 5, and 10, respectively. These findings offer novel insights into the karyotype composition and distribution of 45S rDNA loci in P. sibiricum and P. cyrtonema, contributing to a deeper understanding of chromosomal evolution and genetic differentiation within the genus Polygonatum.