Chromium (Cr) is present in our environment as a toxic pollutant, which needs to be removed using phytoremediation technology. In present study, two transgenic cotton cultivars (J208, Z905) and their hybrid line (ZD14) were used to explore their Cr uptake and tolerance potential using multiple biomarkers approach. Four different levels of Cr (CK, 10, 50, and 100 μM) were applied. Cr caused a significant reduction in root/shoot length, number of secondary roots, and root fresh and dry biomasses at 100 μM. Cr accumulated more in roots and was found higher in hybrid line (ZD14) as compared with its parent lines (J208, Z905) at all Cr stress levels (10, 50, and 100 μM). Cr translocation was less than 1 in all cultivars. Ultrastructural studies at 100 μM Cr showed an increase in number of nuclei and vacuoles and presence of Cr dense granules in dead parts of the cell (vacuoles/cell wall). Malondialdehyde (MDA), hydrogen peroxide (H2O2), total soluble proteins, superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR) as a whole were upregulated with elevated levels of Cr. Higher Cr uptake by roots, accelerated metabolism, and Cr sequestration in dead parts of the cell indicate that these cotton cultivars can be useful for Cr accumulation and tolerance.