Although the photosynthetic responses of sugarcane plants to environmental stresses are well documented, the biomass accumulation and the dynamic of carbohydrate reserves under simultaneous exposure of roots to low temperature and drought are not known. This work aims to investigate the effect of water deficit and low substrate temperature stresses, occurring alone or in combination, on the sugarcane IACSP94-2094, a drought-tolerant genotype. As our hypothesis, we assume that this genotype is also tolerant to low substrate temperature, since low temperatures and water deficit occur simultaneously under field conditions. The water deficit alone or in combination with low substrate temperature caused reductions in leaf water potential and CO2 assimilation, which was not observed in plants subjected only to low substrate temperature. The leaf concentration of non-structural carbohydrates, sucrose and starch increased in plants under root chilling. In plants subjected to water deficit, we noticed decreases in leaf starch concentration. The root stresses caused an increase in the total soluble sugar concentration and reduction in starch concentration in sugarcane roots. As the plant biomass accumulation was not affected, even with the impairment of root growth under low substrate temperature, we conclude that the sugarcane genotype IACSP94-2094 presents evidence of tolerance to low root temperature. The maintenance of plant growth was likely associated with the breakdown of leaf and root reserves of starch.