Arundo donax L. (Poaceae), giant reed, is a rhizomatous shrubby grass that is cultivated in subtropical and warm temperate regions for a multitude of uses. Recently, it has been identified as a leading sustainable, non-food crop for lignocellulosic biofuels due to its low ecological and agronomic demands. Lack of cold hardiness may limit its diffusion into colder areas of the transition zone. The objectives of this study were to (i) quantify freeze tolerance (LT50) of cold-acclimated and non-acclimated Arundo donax L. plants using reproducible, controlled environment conditions, (ii) determine the effect of prolonged exposure to freeze stress on tolerance by keeping plants at a constant, sublethal temperature and (iii) study the relationship of non-structural carbohydrates (total soluble sugars, glucose, fructose and sucrose) and proline accumulation with cold hardiness. In vitro-propagated plants of the Honduran and Hungarian ecotypes of Arundo donax L. were chosen for this study. Cold acclimation treatment was imposed for 1 week using a controlled environment chamber set at 10°C and with a 12-h photoperiod of 200 μmol m−2 s−1 photosynthetically active radiation. Freeze tolerance ranged from −12·8°C (Honduran) to −16·4°C (Hungarian ecotype). In all the organs analysed, total soluble sugars significantly increased during cold acclimation, with concentrations between 1·8- and 4·7-fold higher than in non-acclimated plants. The higher concentrations of sugars and proline in cold-acclimated plants were positively associated with enhanced giant reed freeze tolerance (2·9°C lower). Our results confirm that during cold acclimation, metabolic changes related to increased freezing tolerance occur in giant reed.