A new split-root system was used to simulate non-uniform salt, drought or nutrient deficiency stress in the root zone, in which the root system was divided into two or more equal portions. Here, we established a split-root system by grafting of cotton seedlings. In contrast to the conventional split-root, the main roots of the new system remained intact, which provided a better system for studying cotton response to unequal treatment in the root zone. The new system was suitable for plant growth in nutrient solution and the two root systems can fully be immerged in the nutrient solution.