Background: Artemisia argyi is an economically important medicinal plant with in the genus of Artemisia. However, the lack of the molecular markers hinders the understanding of genetic diversity and cultivar discrimination. Therefore, the main objectives of this study were to characterize the SSRs of Artemisia argyi in the reference genome, and to developed polymorphic SSRs for cultivar discrimination and population genetic studies.BRMethods and Results: Novel 100 simple sequence repeat (SSR) markers were designed based on the whole-genome sequence data of Artemisia argyi, and selected 78 polymorphic markers were selected. Upon the assessment of the five accessions with the full set of 78 SSR, the major allele frequency (MAF) ranged from 0.20 to 1, with an average of 0.36. In addition, the number of alleles per locus ranged from 2 to 9 alleles with a mean value of 5.2 alleles per locus. The polymorphism information content (PIC) values ranged from 0.27 to 0.87 with a mean PIC value of 0.69.BRConclusions: The Artemisia argyi alleles were detected in 5 accessions, among them six primers (argyi-gSSR003, argyi-gSSR014, argyi-gSSR035, argyi-gSSR036, argyi-gSSR093, argyigSSR094) produced five cultivar-specific loci, therefore very promising for cultivar identification, genetic diversity and breeding studies in Artemisia argyi.