Abstract: Duckweed, a small floating aquatic plant, belongs to the monocotyledonous family of Lemnaceae. Duckweed consists of 5 genera and 36 species with a wide distribution in the natural environment. The genetic diversity of duckweed affects adaptability to different natural environments. which is important to evaluate the evolutionary potential and survival pressure of species. Guizhou Province has a subtropical humid monsoon climate area with rich duckweed germplasm resources. To explore the evolutionary potential and survival pressure of duckweed, 41 duckweed ecotypes were collected from 9 cities in Guizhou Province for genetic diversity analysis. The chloroplast atpF-atpH spacer sequence and rpS16 intron sequence were used for molecular biological analysis, and NJ phylogenetic Tree was constructed by MEGA 6.0 software to determine the species classification of samples. Morphological identification showed that 33 ecotypes belong to Lemna, 6 ecotypes belong to Spirodela, and 2 ecotypes belong to Landoltia. Molecular biological showed that 41 ecotypes were clustered into 4 species, including Lemna aequinoctialis, Lemna minor, Spirodela polyrhiza, and Landoltia punctata. The haplotype diversity of the atpF-atpH spacer sequences and rpS16 intron sequences were 0.98700 and 0.64700, the population mutation rates were 0.15380 and 0.14334, nucleotide differences per Kb were 47.20000 and 61.72200 and nucleotide diversity were 0.08725 and 0.09158, respectively. The results provide a basis for genetic relationship analysis of duckweed, and lay a foundation for species identification and resource utilization of plants in subtropical humid monsoon climate area.