To investigate the distribution of phytoplankton as well as their relationship with environmental factors,water samples from different layers were collected in the river and center regions of Xin'anjiang Reservoir.Tempo-ral and vertical variations of phytoplankton were detected using microscopy and Illumina sequencing technique based on eDNA.The results showed that the phytoplankton in the reservoir were mainly composed of Bacillariophy-ta,Chlorophyta,Cryptota,and Cyanobacteria.The dominant genera at Jiekou section site in the river area of the reservoir were Cyanobium(8.3%-37.5%)and Synechococcus(7.6%-30.3%)in winter,while Cyclotella(29.3%)and Cryptomonas(16.2%)were dominant in spring.The dominant genera of the section in front of dam in winter and spring were Cyanobium(16.0%-60.6%)and Synechococcus(7.8%-33.0%).At different depths,the relative abundance of Chlorella at 5 m depth was 15.2%,which was significantly lower than that in other water layers.The relative abundance of phytoplankton Cyanobium at the 5 m depth(50.7%)was significantly higher than that in the other two layers(25.3%and 43.2%,respectively).Chlorophyll a,water temperature,chemical oxygen demand(CODMn)and total nitrogen were the environmental factors affecting the composition and distribution of planktonic communities.Our findings suggested that eDNA-based technique has great potential in analyzing phyto-plankton biodiversity in reservoirs,including prokaryotic algae and eukaryotic algae.