| Summary: | <i>Sanguisorba</i>, commonly known as burnet, is a genus in the family Rosaceae native to the temperate regions of the Northern hemisphere. Five of its thirty species are distributed in Korea: <i>Sanguisorba officinalis</i>, <i>S. stipulata</i>, <i>S. hakusanensis</i>, <i>S. longifolia</i>, and <i>S. tenuifolia</i>. <i>S. officinalis</i> has been designated as a medicinal remedy in the Chinese and Korean Herbal Pharmacopeias. Despite being a valuable medicinal resource, the morphological and genomic information, as well as the genetic characteristics of <i>Sanguisorba</i>, are still elusive. Therefore, we carried out the first comprehensive study on the floral micromorphology, palynology, and complete chloroplast (cp) genome of the <i>Sanguisorba</i> species. The outer sepal waxes and hypanthium characters showed diagnostic value, despite a similar floral micromorphology across different species. All the studied <i>Sanguisorba</i> pollen were small to medium, oblate to prolate-spheroidal, and their exine ornamentation was microechinate. The orbicules, which are possibly synapomorphic, were consistently absent in this genus. Additionally, the cp genomes of <i>S. officinalis</i>, <i>S. stipulata</i>, and <i>S. hakusanensis</i> have been completely sequenced. The comparative analysis of the reported <i>Sanguisorba</i> cp genomes revealed local divergence regions. The nucleotide diversity of <i>trnH-psbA</i> and <i>rps2-rpoC2</i>, referred to as hotspot regions, revealed the highest pi values in six <i>Sanguisorba</i>. The <i>ndhG</i> indicated positive selection pressures as a species-specific variation in <i>S. filiformis</i>. The <i>S. stipulata</i> and <i>S. tenuifolia</i> species had <i>psbK</i> genes at the selected pressures. We developed new DNA barcodes that distinguish the typical <i>S. officinalis</i> and <i>S. officinalis</i> var. <i>longifolia</i>, important herbal medicinal plants, from other similar <i>Sanguisorba</i> species with species-specific distinctive markers. The phylogenetic trees showed the positions of the reported <i>Sanguisorba</i> species; <i>S. officinalis</i>, <i>S. tenuifolia</i>, and <i>S. stipulata</i> showed the nearest genetic distance. The results of our comprehensive study on micromorphology, pollen chemistry, cp genome analysis, and the development of species identification markers can provide valuable information for future studies on <i>S. officinalis</i>, including those highlighting it as an important medicinal resource.
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