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According to the linear elastic fracture mechanics and the virtual work principle, the elemental stiffness matrix of the diagonally cracked RC beam is derived, and the frequencies and modes of the diagonally cracked beam are obtained. The experiment is executed to verify the elemental stiffness matrix. It is given from the experimental result that frequencies would be lower after crack happen, and the influence of higher frequencies is more obviously than influence of the lower frequencies for crack, which is matched with the fact. So the experimental result is reliable. The built elementary stiffness matrix is used to calculate the frequencies and modes of the experimental beams. The maximum relative error of frequencies of calculating and experimental test is controlled in the 2.79%, and the MAC values between calculated mode shapes and experimental mode shapes are all higher than 0.9. The values of calculating with the elementary stiffness matrix built are matched well with the experimental values. So the stiffness matrix of RC beam element with a diagonal crack is applicable in calculation.