Abstract: Environmental DNA metabarcoding targeting the 18S rDNA V9 region was applied to 106 samples collected from 15 stations in the central South China Sea in June 2024. The samples covered mesoscale eddy-influenced and reference areas and were used to investigate the community composition, spatial distribution, diversity patterns, and relationships with environmental factors of pelagic Chlorophyta. The sampling stations represented cyclonic eddy (CE), anticyclonic eddy (ACE), eddy edge, and reference areas. Sequence annotation identified a total of 7 classes and 21 genera of pelagic Chlorophyta, together with an evolutionary lineage without formal taxonomic description, Prasinophyte clade Ⅸ. Chloropicophyceae (Prasinophyte clade VII) was the overwhelmingly dominant group, whereas Prasinophyte clade Ⅸ and Mamiellophyceae were the subdominant groups. The three dominant groups exhibited distinct spatial distribution patterns. The distribution of Chloropicophyceae generally coincided with the deep chlorophyll maximum (DCM) layer, with its dominant genus Chloroparvula showing relatively higher abundance at CE stations and Chloropicon being relatively more abundant at eddy edge stations. Prasinophyte clade Ⅸ was mainly distributed in warm, nutrient-depleted surface waters and showed relatively higher abundance at ACE stations. Mamiellophyceae exhibited relatively high abundance around the 100 m layer, with its dominant genera Bathycoccus and Ostreococcus showing higher relative abundances at reference and CE stations, respectively. Diversity analyses showed that the DCM layer exhibited higher richness and Shannon diversity, whereas deeper waters were characterized by higher community Pielou evenness. Under different eddy conditions, Chlorophyta communities at CE stations showed higher Shannon diversity and Pielou evenness from the surface to the DCM layer, and richness in the DCM layer was also higher at CE stations. Community structure analyses further indicated that mesoscale eddies significantly affected Chlorophyta communities in the upper and middle water layers. Their influence was weaker in deeper waters, with the most pronounced effects observed at CE stations. This study identified the core Chlorophyta groups in the central South China Sea at both class and genus levels and characterized the spatial distribution patterns of pelagic Chlorophyta under different environmental conditions. These findings enrich current knowledge of the biodiversity and ecological distribution of picoeukaryotic phytoplankton in the South China Sea and provide a scientific reference for further understanding the responses of oligotrophic marine ecosystems to physical disturbances.