In order to study the inter-story lateral stiffness damage and identification of ancient wooden structures under strong earthquakes, the inter-story lateral stiffness, lateral displacement response and the sensitivity of displacement to stiffness were analyzed by the shaking table test of an ancient timber frame with a scale ratio 1:3.52. Based on the simplified mechanics model considering the column sliding, the structural state equation and observation equation were deduced. Considering the influence of noise, the lateral stiffness was identified by means of Partial Least Squares-Singular Value Decomposition （PLS-SVD） and Extended Kalman Filter （EKF）. The analysis results show that the ratio of inter-story lateral stiffness gradually decreases with the increase of seismic damage. The damage of column layer is especially sensitive to the increase of lateral displacement. The identified error of the lateral stiffness is about 10% without damage. Under the damage condition, the identified error of the lateral stiffness is about 15%~20%. Through the lateral stiffness identification of Bell Tower of Xi'an, it indicates that the method of PLS-SVD and EKF has high accuracy and good applicability, and can provide a theoretical basis for monitoring the change of lateral stiffness and offering warning before structural collapse.