Pteroceltis tatarinowii, a rare relict plant endemic to China and the sole species of its genus, was both a unique fiber tree and a pioneer species in calcareous soil communities, holding significant economic, cultural, and ecological value. Currently, P. tatarinowii faced population decline and demographic contraction, necessitating immediate conservation-oriented research. This study analyzed the genetic diversity and genetic structure of 210 germplasm resources from 21 wild populations collected from 12 provinces (municipalities) using simplified genome sequencing technology, aiming to provide theoretical basis and practical guidance for the conservation of P. tatarinowii genetic resources. The survey results showed that the habitat of the wild population of P. tatarinowii is fragmented, and its living environment is relatively harsh, with a risk of decline. Sequencing of 210 individuals generated a total of 290.77 Gb of clean data, and a total of 329189 single nucleotide polymorphisms (SNPs) were obtained, among which 217123 polymorphic SNPs. The results of genetic diversity analysis indicated that the species had relatively low genetic diversity, with inbreeding within populations (Ho=0.1992, He=0.2084, Pi=0.2142, Fis=0.0385), and showed mostly moderate genetic differentiation (Fst=0.1201). Gene flow occurred among populations (Nm=1.9002). It was speculated that seed flow was the main form. The gene migration originated mainly from three southwestern populations (GXGL, GXLZ and GZGY) and populations in Anhui Huaibei (AHHB), Shaanxi Huayin (SHXHY), and Jiangxi Hukou (JXHK) to other populations, showing a general gene flow pattern from south to north. The southwestern populations were likely the original populations with two possible population migration paths, and it was speculated that the southwestern region served as a glacial refuge for P. tatarinowii. Population genetic structure analysis divided the 21 P. tatarinowii populations into four subgroups: (1) the northern and central-western region subgroup (NW), (2) the eastern warm temperate region subgroup (ER), (3) the middle and lower reaches of the Yangtze River region subgroup (SR), and (4) the southwestern region subgroup (SW). Additionally, five populations, namely Anhui Huaibei (AHHB), Hubei Macheng (HBMC), Jiangxi Hukou (JXHK), Shandong Qufu (SDQF), and Shaanxi Huayin (SHXHY), were found to contain more unique SNPs and a single genetic background, and thus required the establishment of separate conservation units. The above research results provide theoretical guidance and practical basis for further carrying out the protection work of the genetic resources of P. tatarinowii. It is conducive to promoting the conservation of rare and unique plants in China, thereby contributing to the protection of biodiversity.