Electrodialysis (ED) is receiving increasing attention as promising technology for seawater desalination. However, most of the ED investigations are typically performed using artificial NaCl solutions, while the effect of multivalent ions (such as Mg2+ and Ca2+) on membrane scaling and resistance has been so far overlooked. In this work, we investigate the influence of multivalent ions in seawater on the desalination performance of multistage ED. In particular, natural seawater was used as feed solution, and two different strategies were compared, i.e. by using conventional cation exchange membranes (CEMs), as well as CEMs with preferential removal of multivalent ions. For both CEMs, we found that the removal of calcium and magnesium was higher compared to that of sodium and no effect due to operation at low current density was observed. More magnesium was removed with the multivalent ion permeable CEM. Starting from ~27 g/l (i.e. inlet concentration of the natural seawater source), the upscaled multistage ED system produced a continuous diluate concentration of 1.9 g/l. The system performance was stable over 18 days, with an average energy consumption of 3 kWh/m3, demonstrating the potential of multistage ED seawater desalination.