Cadmium (Cd) tolerance mechanisms in plant are mainly divided into two categories: evasion mechanism and tolerance mechanism. However, due to the complexity of the mechanism of Cd absorption and accumulation in crops, there are still disputes and controversies about Cd toxicity to plants and the mechanism of Cd tolerance in plants. The Cd absorption and accumulation mechanism in edible parts of pepper remains unknown. The present study characterized three pepper cultivars with different cadmium tolerance under cadmium stress. One high-Cd-accumulation type (X55), a medium-Cd-accumulation type (Daguo 99) and a low-Cd-accumulation type (Luojiao 318) were selected to study distribution characteristics of Cd in subcellular fractions of the three pepper varieties as well as expression difference of key Cd accumulation and tolerance genes under different cadmium levels. The results showed that under Cd stress, X55 and Daguo 99 mainly migrated Cd from root to stems and leaves, while Luojiao318 migrated it to the fruit. The Cd concentration in the subcellular fractions of pepper roots, stems, leaves and fruits was as follow: cell wall (F1) > organelle (F2) > cell soluble fraction (F3). The roots, stems and leaf cells of X55 have strong Cd compartmentalization capacity. The fruit cells of Daguo 99 have strong Cd compartmentalization capacity, while the roots of Luojiao318 have strong ability to inhibit Cd absorption. Under Cd stress, HMA1, HMA2 and NRAMP1-6 were up-regulated in roots, stems and fruits of the three varieties. FTP1-2 and FTP1-3 genes were significantly up-regulated in different materials, except the roots of Daguo 99. Under Cd treatment, PCS gene expression of pepper showed an order of that of X55 > Luojiao 318 >Daguo 99. The present study revealed that the cell wall of pepper played an important role in Cd separation and resistance. The difference in Cd accumulation ability of the pepper varieties may be related to differences in main expression sites and expression levels of HMA, NRAMP, FTP and PCS genes. Display omitted •Under Cd stress, Luojiao318 mainly migrated Cd to fruit.•Cd restricted to the cell wall is the detoxification mechanism exposed to Cd.•PCS gene played an important role in Cd detoxification mechanism of pepper.•Higher expression of NRAMP2, 3 &6 in fruit maybe the reason for higher Cd in fruit.