Low temperature during cold acclimation (CA) leads to the accumulation of detrimental reactive oxygen species (ROS) in plant tissues, which are scavenged by antioxidants, such as ascorbate and glutathione. However, there is a lack of studies examining the dynamics of antioxidants throughout CA, deacclimation (DEA), and reacclimation (REA) in winter wheat. Six winter wheat genotypes were selected to assess the effect of CA, DEA, and REA on the concentrations of ascorbate and glutathione in leaf and crown tissues under two CA temperature treatments. Higher levels of total, reduced, and oxidised ascorbate were determined in leaves, whereas crowns accumulated higher concentrations of nicotinamide adenine dinucleotide (NAD+) after CA, DEA, and REA. Constant low temperature (CLT) during CA led to higher contents of ascorbate and glutathione in both tissues at all stages of acclimation, in comparison with prolonged higher low temperature (PHLT). The concentrations of antioxidants increased after CA, tended to decrease after DEA, and returned to CA levels after REA. Significant positive correlations between freezing tolerance (FT) and antioxidants were only determined under the CA at CLT treatment, thus, affirming the negative effect of PHLT during CA on the FT of winter wheat.