Background <p>The accurate identification of herbal medicine is a prerequisite for safe and effective use. DNA barcoding has been widely used for the identification of herbal medicine. However, choosing and refining a suitable barcode is complex in plants. The genus <i>Lysimachia</i> includes over 180 species and is widely used as herbal medicine in China and elsewhere. Species identification within this genus based on morphological, microscopic, or chemical characters is often challenging. In this study, 44 accessions representing 17 putative species of <i>Lysimachia</i> from southern China were sequenced using genome skimming. Whole plastome and nrDNA sequences were successfully recovered from these data. Combining 13 published plastomes of <i>Lysimachia</i>, the discrimination power of standard barcodes, along with plastome and hypervariable sequences from the plastome were compared across 57 plastomes from 22 putative species.</p> Results <p>Standard barcodes have limitation in providing accurate species level identification; however, they can facilitate the rapid classification of unknown samples at the generic level. The plastomes exhibit a highly degree of conservation in the structure and gene content, yet they successfully identify all studied taxa. Five highly variable loci from plastomes were identified: <i>petN</i>-<i>psbM</i>, <i>ycf1</i>, <i>rpl22</i>, <i>trnK</i>-<i>rps16</i>, and <i>ndhC</i>-<i>trnV</i>. While these regions improved the discrimination power compared to standard barcodes, they still could not yield accurate identification of all taxa studied. However, when combined with ITS, <i>ycf1</i> provides 100% successful species identification.</p> Conclusions <p>We recommend that the use of whole plastome and the combination of <i>ycf1</i> + ITS as accurate and effective barcodes for species identification in <i>Lysimachia</i>. This study also highlights the potential for effective use of genome skimming data.</p>