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Flow simulation over a dune requires the proper input of roughness coefficients. This study analyzed a numerical simulation of open-channel turbulent flow over two-dimensional fixed dunes to reveal the effect of roughness on the dune bottom, and to determine the optimized combination of the turbulence scheme and the roughness height formula. The most appropriate roughness values and turbulence models were applied using Reynolds-averaged Navier–Stokes models. Seven methods were chosen to estimate the bed roughness properties at the inlet boundary section. The results of all cases calculated with the OpenFOAM toolbox were compared with laboratory experimental data for model validation. The performances of all bed roughness variations were evaluated according to the stream-wise and depth-wise directions with nondimensional values. Consequently, it was revealed that the combination of bottom roughness length scale at the inlet boundary and the k-ω shear-stress transport (SST) model was the most suitable for the flow separation zone and turbulent properties near the channel bottom.