A rhizobox experiment was conducted to compare the differences of soil potassium (K) distribution and absorption between two cotton (Gossypium hirsutum L.) genotypes under drought and K-deficit conditions. Treatments included two levels of water (drought and optimum soil moisture: 25% and 35% volumetric water content) and K fertilizer rates (0 and 0.48 g potash kg^–1 soil) applied to two cotton genotypes (namely HEG and LEG). Both the genotypes showed significant differences in total K accumulation without exogenous K addition. After absorption, soil content of the readily available potassium (RAK) decreased rapidly. This promoted the conversion of the mineral K into slowly available potassium (SAK). Drought significantly decreased the cotton growth and K use efficiency, and thereby reduced the effect of K fertilizer. Consequantly, the contents of RAK and SAK were greatly increased. However, K bioavailability was decreased under water stress conditions. Differences in root parameters and soil microorganisms between two cotton genotypes were significantly increased and had marked relations with available soil K contents. This study provides important information for understanding the mechanism of K use efficiency, especially under water and K stress.