BACKGROUND: Arctium lappa L. is a medicinal edible homologous plant, commonly known as burdock or bardana, which belongs to the Asteraceae family and is abundant all over the world. Genetic diversity assessment is essential for A. lappa germplasm resource conservation and breeding. The assessment techniques include morphological, biochemical, and DNA marker analysis. However, the limited number of available DNA markers is insufficient to conduct related genetic diversity assessment studies. RESULTS: In this study, we conducted RNA sequencing of the A. lappa cultivar 'Yanagikawa Ideal' and developed SSR markers to characterize the genetic diversity and population structure of 56 A. lappa accessions and 8 wild relative accessions. A total of 4,851 simple sequence repeats (SSRs) loci were identified. The proportions of mono-, di- and tri-nucleotide repeat motifs were 30.40%, 21.50% and 33.10%, respectively. We developed and verified the reliability of 28 SSR core primer pairs through electronic polymerase chain reaction (ePCR) and the PCR amplification process. The polymorphism information content (PIC) values of the 28 SSR core primer pairs ranged from 0.246 to 0.848, with 14 pairs of SSR primers displaying high polymorphism (PIC >
0.5). The 28 SSR core primer pairs showed 100% mobility in Arctium tomentosum Miller and 96.43% mobility in Synurus deltoides (Aiton) Nakai, indicating their high versatility. The average Shannon information index (I) was 1.231, and the average observed heterozygosity (Ho) was 0.132, the average expected heterozygosity (He) was 0.564. The 64 accessions were divided into three clusters at a genetic distance of 0.558. AMOVA analysis shows 83% genetic variation within populations and 17% among populations, highlighting implications for conservation and breeding strategies. CONCLUSION: Our study provides 28 newly high-quality SSR markers to enhance genetic resource conservation and breeding programs for A. lappa, as well as to support comparative genomics and cross-species breeding strategies for related species.