Tobacco (Nicotiana tabacum L.) is a potential phytoremediator that can reduce soil cadmium (Cd) contamination. Pot and hydroponic experiments were conducted to investigate the difference in absorption kinetics, translocation patterns, accumulation capacity, and extraction amounts between two leading tobacco cultivars in China. We studied the chemical forms and subcellular distribution of Cd in the plants to understand the diversity of the detoxification mechanism of the cultivars. The concentration-dependent kinetics of Cd accumulation in leaves, stems, roots, and xylem sap for cultivars Zhongyan 100 (ZY100) and K326, fitted well with the Michaelis-Menten equation. K326 exhibited high biomass, Cd tolerance, Cd translocation, and phytoextraction abilities. The acetic acid, sodium chloride, and water-extractable fractions accounted for > 90% of Cd in all ZY100 tissues but only in K326 roots and stems. Moreover, the acetic acid and NaCl fractions were the predominant storage forms, while the water fraction was the transport form. The ethanol fraction also contributed significantly to Cd storage in K326 leaves. As the Cd treatment increased, more NaCl and water fractions were found in K326 leaves, while only NaCl fractions increased in ZY100 leaves. For subcellular distribution, > 93% Cd proportions were primarily stored in both cultivars' soluble or cell wall fraction. The proportion of Cd in the cell wall fraction of ZY100 roots was less than that of K326, while that proportion in the soluble fraction in ZY100 leaves was higher than in K326 leaves. These findings demonstrate that Cd accumulation patterns, detoxification, and storage strategies differ between the cultivars, providing a deeper understanding of Cd tolerance and accumulation mechanism in tobacco plants. It also guides the screening of germplasm resources or gene modification to improve the Cd phytoextraction efficiency of tobacco.