Biomass accumulation, physiological and biochemical responses of sugarcane to ozone (O3) were evaluated in two genotypes (IACSP94-2094 and IACSP95-5000) in a realistic free-air O3 exposure system. Our aims were to investigate the mechanisms involved in O3 detoxification and verify whether such mechanisms are genotype-dependent. Plants were exposed to ambient air (Amb.) and ambient air plus 40% increase in O3 concentration (x1.4) for 90 days. Plants at x1.4 O3 showed reductions in leaf gas exchange and biomass production with consistent differences between the genotypes. Photosynthesis of IACSP95-5000 was more sensitive to elevated O3 as compared to IACSP94-2094. As a primary response, IACSP94-2094 showed reductions in stomatal conductance due to elevated O3, thus avoiding oxidative stress and maintaining CO2 assimilation for longer. As a consequence, IACSP94-2094 produced more biomass, and stalks had more soluble sugar concentration than IACSP95-5000. Leaf concentrations of O2− and H2O2 in plants exposed to x1.4 O3 were not enhanced, with such unexpected result suggesting that sugarcane plants have an effective antioxidant system to avoid O3-induced damage. In both genotypes, increases in ascorbate peroxidase activity was directly related to decreases in leaf H2O2 concentration. In IACSP94-2094, however, catalase pathway was also stimulated for detoxifying H2O2. We concluded that sugarcane sensitivity to O3 is genotype-dependent and then it is possible to reduce the deleterious effects of high O3 levels on sugarcane physiology and biomass production by growing tolerant genotypes in polluted areas.