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Existing studies have proved that interfaces between the prefabricated layer and the cast‐in‐place layer significantly affect the static performance of prefabricated concrete structures using the wet connection method, but little has been known about the fatigue performance, especially at high load levels. This study investigated the fatigue behavior of prefabricated composite concrete beams under negative bending moment induced by the high‐stress level cyclic load. Fifteen composite T‐beams with three different designed sections were tested. The failure mode, cracking and deflection behavior, steel strain development, and the new‐old concrete interfacial deformation were recorded and analyzed. It was found that the stress redistribution occurred with the debonding cracks generated at the new‐old concrete interface, causing the degradation of the static and fatigue performance. Setting interfacial shear keys could prevent such debonding failure at static and low cyclic loading levels. Meanwhile, the corresponding mechanisms were verified through cross‐sectional analysis. [ABSTRACT FROM AUTHOR]