This study was conducted on well-watered citrus to determine changes in water relations during cold acclimation independent of drought stress. Potted sweet orange and Satsuma mandarin trees were exposed to progressively lower, non-freezing temperatures down to 10/4 °C, light/dark temperatures, respectively, for 9 weeks in environmental growth chambers to promote cold acclimation. The trees were watered twice daily and three times on the day water relations data were collected to minimize drought stress. Although soil moisture was higher and non-limiting for plants in the cold than in the warm chamber, cold temperatures promoted stomatal closure, higher root resistance, lower stem water potential (Ψstem), lower transpiration, and lower leaf ψS. Leaf relative water content (RWC) was not different for cold-acclimated trees compared with the controls. Cold acclimation promoted stomatal closure at levels only observed in severely drought-stressed plants exposed to warm temperatures and where Ψstem and RWC are typically much lower than what was found in this study. Ψstem continued to decline the last 4 weeks of the experiment although air temperature, leaf ψS, RWC, stomatal conductance (gS), and transpiration were constant. The results of this experiment indicate that water relations of citrus during cold acclimation vary from those known to occur as a result of drought stress, which have implications for using traditional measures of plant water status in irrigation scheduling during winter.