Duckweed, a family of aquatic plants, has the characteristics of fast growth and reproduction, strong nitrogen and phosphorus absorption capacity, and high protein and starch content. Therefore it has great potential in feed and bioenergy production. However, the information of duckweed germplasm collection and diversity analysis in the middle reaches of Changjiang River has not been systematically reported. In this study, 112 duckweed germplasm were collected from 34 representative natural aquatic habitats in three provinces of the middle reaches of the Changjiang River. These germplasm resources were classified into 4 genera and 6 species by morphological and molecular biological identification. Lemna contained 3 species and 34 accessions. Spirodela, Landoltia and Wolffia contained only 1 species, and their collected germplasm number was 33, 19 and 6, respectively. Genetic diversity analysis showed that the number of haplotypes of their AtpF-AtpH spacer sequences and rpS16 intron sequences were 11 and 18, the haplotype diversity was 0.748 and 0.892, the population mutation rates were 0.03439 and 0.03507, nucleotide differences per Kb were 31.386 and 61.263 and nucleotide diversity was 0.04713 and 0.06623, respectively. This study plays a key role for the utilization of duckweed in the middle reaches of the Changjiang River.