Abstract: Maoli Lake is located in the lower reaches of Lishui River, one of the four major river systems in Hunan Province. As the largest stream lake in Hunan Province with rich fish resources, no systematic research has been carried out on its fish diversity and distribution till now. Therefore, understanding the status quo and distribution of fish diversity is of great significance for the conservation of local aquatic biodiversity. Based on a survey of 19 sampling sites in the spring of 2023, the fish diversity and distribution pattern in East Maoli Lake, West Maoli Lake and the streams entering them were investigated using eDNA (environmental DNA) method. The results show that there were 47 fish species detected in the study area, belonging to 7 orders, 14 families and 36 genera, of which 80.85% belong to Cypriniformes and Perciformes. The dominant species were Micropercops swinhonis (26.07%), Megalobrama amblycephala (15.47%) and Cyprinus carpio (11.67%). The rare fish species were Macropodus chinensis and Acheilognathus hypselonotus. The average Shannon-Wiener's diversity index was 2.38. The fishes in Maoli Lake are mainly omnivorous and carnivorous, and there are more fish species in the lower layer of the water in terms of community structure. The results of univariate ANOVA show that there were significant differences in Pielou Evenness Index (P < 0.05) and Fish Communities (P < 0.001) between lakes and permanent streams. Moreover, the lake areas had more abundant fish species and higher ecosystem stability. The redundancy analysis show that the distribution pattern were mainly influenced by pH and temperature of the aquatic environment. This study can provide basics for the application of eDNA technology to the survey of fish resources and provide a reference for the conservation of aquatic biodiversity in Maoli Lake and permanent streams entering the lake.