The aim of this study was to assess seasonal variation in the food-web structure of fish assemblages in the East (two sites) and the South (one site) Seas of Korea, and to compare the isotopic niche areas between the regions. To do this, we analyzed the community structures and the δ13C and δ15N values for fish assemblages, and their potential food sources collected during May and October 2020. There were spatial differences in the diversity and dominant species of fish assemblages between the two seas. The fish assemblages in the South Sea had relatively wide ranges of δ13C and δ15N (−22.4‰ to −15.3‰ and 7.4‰ to 13.8‰, respectively) compared to those (−22.1‰ to −18.0‰ and 9.8‰ to 13.6‰, respectively) in the East Sea. The δ13C and δ15N values of suspended particulate organic matter, zooplankton, and fish assemblages differed significantly among sites and between seasons (PERMANOVA, p < 0.05, in all cases). Moreover, isotopic niche indices were relatively higher in the South Sea compared to those in the East Sea. Such differences in food-web characteristics among sites are likely due to the specific environmental effects (especially, major currents) on the differences in the species compositions and, therefore, their trophic relationships. Overall, these results allow for a deeper understanding of the changing trophic diversity and community structure of fish assemblages resulting from climate variability.