This study analyzed the structural characteristics and influencing factors of fish assemblages according to lake size (small, medium, large) based on fish survey data from 90 lakes designated under the National Aquatic Ecosystem Health Monitoring Program in Korea. From surveys conducted between 2022 and 2024, a total of 107 fish species belonging to 32 families were recorded. The dominant species was the bluegill (Lepomis macrochirus, relative abundance [RA]: 21.2%), followed by Hemiculter eigenmanni as the subdominant species (RA: 13.6%). Statistical analysis based on lake size revealed significant differences across eight ecological indicators including species richness, abundance, diversity, evenness, richness, dominance, number of endemic species, and number of exotic species according to lake size. These indicators tended to increase with lake size. While this pattern may be initially attributed to positive factors associated with larger lakes, such as greater habitat heterogeneity and food resource diversity, it is considered that the primary factor influencing these results is the difference in the number of sampling sites per lake. Since sampling sites were designated based on the national water quality monitoring network, they are considered representative of each lake’s environmental conditions. Bray-Curtis similarity and SIMPER analyses identified patterns in assemblage similarity and the key contributing species for each size group. This study provides empirical evidence that demonstrates the influence of lake size on fish assemblage composition and structure and highlights the necessity of incorporating lake size and typology into fish-based assessments of lentic ecosystem health.