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Monitoring and characterizing species biodiversity is essential for germplasm preservation, academic studies, and various practical applications. Duckweeds represent a group of tiny aquatic plants that include 36 species divided into 5 genera within the Lemnaceae family. They are an important part of aquatic ecosystems worldwide, often covering large portions of the water reservoirs they inhabit, and have many potential applications, including in bioremediation, biofuels, and biomanufacturing. Here, we evaluated the biodiversity of duckweeds in Ukraine and Eastern China by characterizing specimens using the two-barcode protocol with the chloroplast <i>atpH–atpF</i> and <i>psbK–psbI</i> spacer sequences. In total, 69 Chinese and Ukrainian duckweed specimens were sequenced. The sequences were compared against sequences in the NCBI database using BLAST. We identified six species from China (<i>Spirodela polyrhiza</i>, <i>Landoltia punctata</i>, <i>Lemna aequinoctialis</i>, <i>Lemna minor</i>, <i>Lemna turionifera</i>, and <i>Wolffia globosa</i>) and six from Ukraine (<i>S. polyrhiza</i>, <i>Lemna gibba</i>, <i>Lemna minor</i>, <i>Lemna trisulca</i>, <i>Lemna turionifera</i>, and <i>Wolffia arrhiza</i>). The most common duckweed species in the samples from Ukraine were <i>Le. minor</i> and <i>S. polyrhiza</i>, accounting for 17 and 15 out of 40 specimens, respectively. The most common duckweed species in the samples from China was <i>S. polyrhiza</i>, accounting for 15 out of 29 specimens. <i>La. punctata</i> and <i>Le. aequinoctialis</i> were also common in China, accounting for five and four specimens, respectively. According to both <i>atpH–atpF</i> and <i>psbK–psbI</i> barcode analyses, the species identified as <i>Le. aequinoctialis</i> does not form a uniform taxon similar to other duckweed species, and therefore the phylogenetic status of this species requires further clarification. By monitoring duckweeds using chloroplast DNA sequencing, we not only precisely identified local species and ecotypes, but also provided background for further exploration of native varieties with diverse genetic backgrounds. These data could be useful for future conservation, breeding, and biotechnological applications.