The study revealed genetic diversity and moderate differentiation among the R. palmatum complex within the top-geoherb area. RNA-seq-derived SNP datasets hold the potential to trace geographic origins of the core germplasm. The Rheum palmatum complex, the source plant of rhubarb, has been widely used for centuries due to its diverse functions in clinical treatments. However, the wild resources of rhubarb are currently declining and even facing depletion. Therefore, revealing the genetic background of the R. palmatum complex within the top-geoherb area is important for the efficient utilization and conservation of its wild resources. In this study, population transcriptomic analyses were conducted to assess the genetic diversity and gene expression diversity of different populations in the R. palmatum complex within the top-geoherb area. Candidate single nucleotide polymorphisms (SNPs) were identified as specific molecular markers for tracing the origin of the R. palmatum complex from various top-geoherb areas. Based on the reference genome, a total of 30,480 transcripts and 100,966 SNPs were generated across 82 individuals from 17 populations of the R. palmatum complex. Moderate genetic differentiation was detected for the two genetic lineages of the R. palmatum complex derived from the top-geoherb area. Fourteen genes encoding key enzymes were differentially expressed between two genetic lineages. Besides, 26 specific SNPs located on the genes involved in the biosynthesis of the active components were screened out, and these SNPs were highly differentiated between 2 genetic lineages. A large-scale reference-based assembly transcriptome of the R. palmatum complex from the top-geoherb area provided insights into the genetic divergence and expression differentiation of genetic lineages. The results not only help to understand the genetic background of the R. palmatum complex in the top-geoherb area but also contribute to future genetic conservation and directive breeding of rhubarb.