Abstract: In order to explore the genotype difference mechanism of Cd vacuole interception in tobacco root systems, hydroponic seedling experiments with different Cd treatments were carried out to study the differences of root system Cd chelating ability and related genes expression between two tobacco genotypes (K326 and N. rustica). The results showed that: 1) After Cd treatments, GSH decreased, while phytochelatins (PCs) increased. Comparing with the control, the contents of total non-protein thiol (NPT) and PCs in the roots of K326 and N. rustica in the treatment of 1 μmol/L Cd increased by 45.89%, 57.75% and 55.58%, 84.00%, while GSH content decreased by 10.70% and 62.09%, respectively. The change of GSH and PCs caused by Cd treatment was more significant in N. rustica than in K326. 2) Cd treatment promoted Cd in roots transforming from exchangeable state into chelated state, and promoted PCs transforming into high-molecular-weight bound state (HMW). Notably in N. rustica. 3) In the control, HMW and LMW each accounted roughly for half of the root system chelates in K326 or N. rustica. However, induced by the treatment of 1 μmol/L Cd, HMW accounted for 77.43% of root chelates in K326, and almost all Cd was in the form of HMW state in N. rustica. Under Cd inducing conditions, the transition ability of Cd to chelated state and the binding ability were significantly higher in N. rustica than in K326, it indicated that the difference of roots'Cd chelating ability was one of the reasons causing the difference of Cd accumulation between K326 and N. rustica. 4) PCS1 expression in the roots of N. rustica was highly sensitive to Cd, and increased by 257.33% in the treatment of 1 μmol/L Cd; while PCS1 expression in K326 was not affected significantly by Cd treatment.