Understanding trophic interactions in food webs is crucial to revealing the transfer of substances and energy from primary food sources to consumers in aquatic ecosystems. We hypothesize that the trophic structure of consumers can be significantly affected by primary food sources (pelagic, benthic, and littoral sources) through complex trophic interactions. This study used stable isotope analysis and Bayesian mixing models to estimate the trophic levels of fish consumers and the contributions of primary food sources in the three sub-lakes (Eastern, Southern, and Western Dongting) of Lake Dongting, which have different physical and chemical parameters of water, fish species diversity, and plankton (phytoplankton and zooplankton) density. Results showed the differences in community structures of fish among sub-lakes. Fish trophic levels were significantly higher in Eastern Dongting than those in the two other areas. The contributions of primary food sources to fishes were as follows: the pelagic source was the main basal food source in Eastern Dongting, and littoral and pelagic sources played equally essential roles in Southern Dongting; fishes in Western Dongting relied on more benthic source to growth than those in the two other regions. This study can fill gaps in our knowledge of the influence of the underlying food available on trophic structure of consumers by exploring the role of primary food sources and making the trophic structure of consumers in the aquatic food web highly complicated and diverse through control of the distribution of primary food sources.