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Positioning accuracy is of great significance for industrial applications. Nevertheless, in actual machining operations, deformation will generate on the endeffector of industrial manipulators under external loads due to the flexibility of actuated joints. In industrial environment, the stiffness identification accuracy of serial manipulators is affected by various measurement errors. However, there is little research on dealing with the inevitable error perturbation. An optimization method and experimental design were proposed for measurement configuration of manipulator stiffness identification. Firstly, κ－1F(A) was adopted as the evaluation criterion of measurement configuration considering comprehensively the influence of manipulator posture and wrench on stiffness identification. On this basis, optimal configurations based on κ－1F(A) were obtained by the appropriate simulated annealing algorithm. The optimized loading was achieved on the basis of the designed multi-directional loading setup. The experimental results showed that compared with the typical evaluation criterion κ－1F(J), the optimal configurations based on κ－1F(A) can better overcome the impact of various measurement errors. After displacement compensation, the end position accuracy was increased by 29.59%, and the maximum end position error was reduced by 32.71% compared with the typical criterion set. The proposed method can be subsequently applied to the stiffness identification of serial manipulators in industrial environment.