Inter-basin water transfer projects, such as the Yellow River to Qingdao Water Diversion Project (YQWD), are essential for addressing water scarcity, but impact local aquatic ecosystems. This study investigates the seasonal characteristics of eukaryotic microbial communities in the Jihongtan Reservoir, the main water-receiving body of YQWD, over a one-year period using 18S rDNA amplicon sequencing. The results showed that the eukaryotic microbial diversity did not exhibit significant seasonal variation ( p > 0.05), but there was a notable variance in the community structure ( p < 0.05). Arthropoda and Paracyclopina , representing the most dominant phylum and the most dominant genus, respectively, both exhibited the lowest abundance during the winter. The Chlorophyta, as the second-dominant phylum, demonstrates its higher abundance in the spring and winter. The Mantel test and PLS-PM (Partial Least Squares Path Modeling) revealed that water temperature (WT), dissolved oxygen (DO), and pH influenced the seasonal dynamic of eukaryotic microbial communities significantly, of which WT was the primary driving factor. In addition to environmental factors, water diversion is likely to be an important influencing factor. The results of the co-occurrence network and robustness suggested that the spring network is the most complex and exhibits the highest stability. Moreover, keystone taxa within networks have been identified, revealing that these key groups encompass both abundant and rare species, with specificity to different seasons. These insights are vital for understanding the seasonal variation of microbial communities in the Jihongtan Reservoir during ongoing water diversions.