The difference in seepage resistance of each layer and interlayer resistance in a multilayer reservoir can directly characterize interlayer interference and its changes during development. However，the present formulas for calculating seepage resistance are featured by complicated computation or low accuracy. On the basis of the Buckley-Leverett theory，a novel calculation method of two-phase（oil-water）seepage resistance is given in this paper for the one-dimensional water flooding process，and its accuracy is verified. According to this method，the calculation process of the interlayer resistance difference during water flooding of a multilayer reservoir is further proposed，which can predict and analyze the changes in the interlayer interference during development. The results demonstrate that the values of seepage resistance calculated by our method agree well with the results of numerical simulation，with the relative error less than 12%. The variation of seepage resistance presents the stages of linear，gradual，and nearly gentle decrease，corresponding to the periods of water-free oil production，medium-high water cut，and ultra-high water cut. The interlayer resistance difference during the water flooding process of the multilayer reservoir reaches a peak after the high-permeability layer enters the period of ultra-high water cut，which can serve as a transition point for adjustment measures. The increase in the permeability difference could rapidly enlarge interlayer interference. Therefore，early control measures are essential for multilayer heterogeneous reservoirs.