Triphenyltin (TPhT) and tributyltin (TBT) remain widely present in various aquatic environments despite restrictions on their use in many countries for many years. The biomagnification of these compounds in the aquatic food web remains controversial. This study reports the bioaccumulation of TPhT and TBT in aquatic animals in the Three Gorges Reservoir (TGR), a deep-water river channel-type reservoir and the largest reservoir in China. We measured TPhT, TBT and their metabolites in 2 invertebrates, 27 fish and the aquatic environment. The logarithmic bioaccumulation factors of TPhT and TBT were 4.37 and 3.77, respectively, indicating that TPhT and TBT were enriched in organisms of the TGR. Both TPhT and TBT concentrations were significantly and positively correlated with trophic level, with trophic magnification factors of 3.71 and 3.63, respectively, indicating that TPhT and TBT exhibited similar trophic enrichment in the freshwater food web of the TGR. The results of health risk assessment showed that although all hazard index (HI) values were <1, more attention should be paid to the health risk to children associated with consumption of aquatic products (HI = 0.67). This study provides powerful evidence of trophic enrichment of TPhT and TBT in a freshwater food web in a deep-water river channel-type reservoir and provides valuable data regarding organotins in aquatic animals in the TGR.