The stratification of sedimentary aquifers introduces spatial variability in hydraulic conductivity, primarily between individual horizontal layers. On larger scales, the vertical heterogeneity enhances hydraulic anisotropy, with the horizontal conductivity typically exceeding the vertical one. In this study, the hydraulic anisotropy of a stratified aquifer is estimated from data of hydraulic tests in which water is sequentially extracted from well sections screened at different depths, and the hydraulic response is measured at various multilevel observation wells. The applicability of the method is demonstrated by field tests in a fluvial gravel aquifer in the Upper Rhine Valley, Germany. A homogeneous anisotropic model, and models with three and five anisotropic layers, are fitted to the measured drawdowns in the steady-shape regime, in which differences in hydraulic head between observation locations do not change over time even though the head values themselves change. The position of the five horizontal layers is based on the lithology of the drilling profile at the pumping-well location. The three-layer model is achieved by merging insensitive or similar layers with sensitive layers. The fits result in estimates of the radial and vertical hydraulic conductivities for all layers of the respective models, which are upscaled to effective parameters over the entire depth in the case of the multilayer models. The homogeneous model shows significantly higher errors than those of the heterogeneous models. The heterogeneous locally anisotropic models not only reveal vertical variability of hydraulic conductivity, but also lead to a three-times larger anisotropy ratio upon upscaling.