A novel in-situ non-flatness measurement method of wafer stage mirrors in a step-and scan lithographic tool is presented. According to the impact of mirror non-flatness on wafer stage′s positioning, the non-flatness of the mirror is expressed as translation compensation and rotation compensation usually. The double frequency laser interferometers are used to measure the horizontal positions and rotations of the wafer stage. The whole measurement is divided into several sequences. Each sequence includes a whole measurement of the valid area of the mirror. The rotation compensation of the mirror is calculated from each measured values of the wafer stage′s rotation. The coarse translation compensation of the mirror is calculated recursively by setting a temporary boundary condition in each sequence. The cubic spline interpolation method and least square method are used to smooth and connect all the coarse translation compensations of the mirror to calculate the accurate translation compensation of the mirror. Experiment results prove that by this method the measurement reproducibility of the translation compensation of mirror is better than 2.957 nm and the measurement reproducibility of the rotation compensation of mirror is better than 0.102 μrad.