| Summary: | Chinese herbal tea, also known as Liang Cha or cooling beverage, is popular in South China. It is regarded as a quick-fix remedy to relieve minor health problems. <i>Hedyotis diffusa</i> Willd. (colloquially Baihuasheshecao) is a common ingredient of cooling beverages. <i>H. diffusa</i> is also used to treat cancer and bacterial infections. Owing to the high demand for <i>H. diffusa</i>, two common adulterants, <i>Hedyotis brachypoda</i> (DC.) Sivar and Biju (colloquially Nidingjingcao) and <i>Hedyotis corymbosa</i> (L.) Lam. (colloquially Shuixiancao), are commonly encountered in the market. Owing to the close similarity of their morphological characteristics, it is difficult to differentiate them. Here, we sequenced the complete chloroplast genomes of the three species of <i>Hedyotis</i> using next-generation sequencing (NGS). By comparing the complete chloroplast genomes, we found that they are closely related in the subfamily Rubioideae. We also discovered that there are significant differences in the number and repeating motifs of microsatellites and complex repeats and revealed three divergent hotspots, <i>rps16-trnQ</i> intergenic spacer, <i>ndhD</i> and <i>ycf1</i>. By using these species-specific sequences, we propose new DNA barcoding markers for the authentication of <i>H. diffusa</i> and its two common adulterants.
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