Purpose: To facilitate the molecular identification of original plants, resolve taxonomic problems and identify standards for ‘Xihuangcao’-based products on the market.Methods: A transcriptomic analysis of two closely related species, i.e., Isodon serra (Maxim.) (IS) and I. lophanthoides (Buch.-Ham. ex D. Don) Hara, was conducted by using the Illumina HiSeq 2500 platform, and expressed sequence tag-derived simple sequence repeat (EST-SSR) markers were developed based on these transcriptomes.Results: In total, 149,650 and 103,221 contigs were obtained, with N50 values of 1,400 and 1,516, from the IS and I. lophanthoides RNA-Seq datasets, respectively. These contigs were clustered into 107,777 and 68,220 unigenes, which were functionally annotated to identify the genes involved in therapeutic components. In total, 14,138 and 11,756 EST-SSR motifs were identified, and of these motifs, 7,453 and 6,428 were used to design primers for IS and I. lophanthoides, respectively. After PCR verification and fluorescence-based genotyping, 24 SSR markers with bright bands, high polymorphism, and single amplification were obtained and used to identify closely related Isodon species/varieties.Conclusion: These data could help herbal scientists identify high-quality herbal plants and provide a reference for genetic improvement and population genetic and phylogenetic studies investigating ‘Xihuangcao’ herbs.Keywords: Xihuangcao, Transcriptome, EST-SSR, Molecular marker
