The study aimed to investigate the effects of heat stress on sugar beet growth and physiology. Two field tests were conducted at temperatures of 42 °C (heat stress) and 23 °C (moderate temperature) in different seasons. The results showed that sugar beet leaves grown under heat stress conditions exhibited higher levels of Malondialdehyde (MDA) and hydrogen peroxide (H2O2) compared to moderate temperature conditions in both seasons. Additionally, the activity of antioxidant enzymes, such as superoxide dismutase (SOD) and ascorbate peroxidase (APX), significantly increased in response to high temperatures. However, catalase (CAT) activity was lower in the second season's high-temperature samples compared to moderate temperature samples. The polypeptide patterns of the high-temperature samples showed the presence of new polypeptides (M.Wts of 123, 110, 103, 77, 51, 39, and 31 kD), while moderate temperature samples did not have these polypeptides. Agronomic traits revealed lower values of root weight, top fresh weight, root fresh yield, top fresh yield, and sucrose% in high-temperature conditions. Furthermore, gross sugar yield was lower, while sugar lost% and α-amino-N concentration were higher in the high-temperature conditions. Multivariate analysis was conducted to examine correlations between biochemical and developmental traits under heat stress.