Abstract: Pulsatilla vulgaris (pasque flower) is a rare and endangered plant species. Using ITS1-5.8S rDNA-ITS2 barcode sequencing and locus-specific NGS of the ITS1 spacer, we studied intraspecific and interspecific polymorphism of 35S rDNA loci and investigated the origin of tetraploid P. vulgaris from a unique Russian population. For the first time, the chromosome number in the karyotype of plants from this population was determined as 2n = 32. It was shown that one of the parental species of this tetraploid is the diploid Pulsatilla patens (2n = 16), while the identity of the second parental species remains unknown, although it appears to be closely related to the diploid Pulsatilla pratensis. © Pleiades Publishing, Ltd. 2026.