Root growth influences plant water adsorption. Biochar amendment has been shown to alter root growth and plant water status; however, the physiological mechanisms are still unknown. To investigate this, we analysed root morphology, anatomy, and leaf water status of two contrasting maize genotypes (osmotic -sensitive KN5585 vs. -tolerant Mo17) after biochar amendment under three soil water conditions. The biochar treatment induced a significant increase in root sap K+ concentration while a decrease in root sap Ca2+ and Na+ concentrations for two maize genotypes compared to the no biochar treatment. For KN5585, the root apoplastic pH was increased with elevated sap K+ concentration in the biochar treatment. However, the lateral root number and length as well as leaf relative water content were decreased in the biochar treatment for KN5585. For Mo17, there were no significant differences in root apoplastic pH, lateral root number and length between the no biochar and biochar treatments. Nevertheless, the root xylem vessel diameter was increased in the biochar treatment, contributing to increased leaf water content for Mo17. We elucidated that, as K+ concentration increased after biochar amendment, the osmotic -sensitive maize genotype increased its root apoplastic pH to inhibit lateral root growth, which deteriorated plant water status; while, the osmotic -tolerant maize genotype enlarged its root xylem vessel to increase water absorption. These results indicate that biochar effects on root growth and plant water status were dependent on maize genotypes during seedling stage.