To address the demand for natural fibers, developing new varieties that are resistant to abiotic stress is necessary. The present study was designed to investigate the physiological and biochemical traits of three varieties of C. capularis (Y49, Y38, and Y1) and four varieties C. olitorius (T8, W57, M33, M18) under low temperature to identify the cold-tolerant varieties and elucidate the mechanisms involved in enhancing cold tolerance. Research findings revealed that the varieties Y49 and M33 exhibited the highest chlorophyll and carotenoid content. Biochemical profiles revealed that varieties Y49 and M33 were found to be able to withstand low-temperature stress by accumulating different enzymatic and non-enzymatic antioxidants, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APx), glutathione (GSH), and phenolics, which participated in reducing the content of malondialdehyde (MDA) and hydrogen peroxide (H2O2) caused by low temperature. Osmolytes compounds, such as total soluble sugar, significantly increased in Y49 and M33; and proline content decreased in all varieties except Y49 and M33 after low-temperature exposure. The rise in these osmolytes molecules can be a defense mechanism for the jute’s osmotic readjustment to reduce the oxidative damage induced by low temperature. Furthermore, PCA and hierarchical cluster analysis distinguished the seven varieties into three separate groups. Results confirmed that group I (Y49 and M33 varieties) were low-temperature tolerant, group II (M18, W57) were intermediate, whereas III groups (Y38, T8, and Y1) were low temperature susceptible. PCA also explained 85.72% of the variance of raw data and clearly distinguished three groups that are similar to the cluster heat map. The study thus confirmed the tolerance of selected varieties that might be an efficient adaptation strategy and utilized them for establishing breeding programs for cold tolerance.