An experiment on hydroponic seedlings was carried out to study the subcellular distribution and chemical forms of Cd in the root system and leaves of two tobacco genotypes (K326 and N. Rustica) with different Cd accumulation pattern at different Cd treatment level (1 and 50 μmol/L). The results showed that: 1) At low level, Cd accumulated in root system and leaves of K326, while accumulated mainly in root system of N. rustica. At high level, Cd mainly accumulated in root system of both varieties. 2) At low level, Cd mainly bound to the soluble fraction in both varieties, which accounted for about 70% of the total Cd content. The Cd retention by cell wall increased significantly in K326 at high Cd level. 3) The binding proportion of Cd in soluble fraction always identical with the Cd content in respective parts, which indicated that the difference of binding ability in soluble fraction might be one of the major factors affecting Cd accumulation in the two genotypes. 4) Ethanol-extractable Cd in root systems of K326 and N. rustica accounted for 72.15% and 71.36% of the total Cd, respectively, it was the main chemical form of Cd in root system. In leaves, Cd mainly existed in the forms of binding to NaCl and deionized water. No significant difference was observed between the chemical forms of Cd in the two genotypes, which indicated that Cd accumulation in tobacco was not relevant to its chemical form.