To understand karyotype variation in eight populations, detailed karyotypes were meticulously established using chromosomal measurements, fluorescence bands, and rDNA FISH signals. The number of 45S rDNA sites varies from one to five pairs per population, with the most common number per karyotype being four pairs. The 45S rDNA locus is predominantly located in the short arms and terminal regions of chromosomes, while the 5S rDNA locus is found mainly in the short arm and the terminal or proximal regions. Populations HBWF, HNXN, HBBD, and HNZX showed a similar distribution of 45S rDNA sites, as did GXTL, HBFC, and SCLS, indicating a close relationship between populations with similar 45S rDNA site distributions. The karyotypes of all studied populations are symmetrical, comprising stable and metastable centromeres or exclusively stable centromeres. Scatter plots of MCA and CVCL effectively distinguish their karyotypic structures. The analysis includes six quantitative parameters (x, 2n, TCL, MCA, CVCL, CVCI). Additionally, the results indicate that PCoA based on these six parameters is a robust method for determining biological karyotype relationships among the eight populations. The chromosome number in Lycoris populations is x = 6-8. Based on the current study and literature, genomic differentiation of these populations is discussed in terms of genome size, heterochromatin, 45S and 5S rDNA sites, and karyotype asymmetry.