Sugarcane (Saccharum spp.) growth and yield decrease in acidic soil (low pH and high Al content) conditions owing to impaired nutrient and water absorption. An efficient method to evaluate acid tolerance needs to be developed to utilize Erianthus arundinaceus to improve acid tolerance in sugarcane. Herein, we performed a root electrolyte leakage assay to quantitatively evaluate and compare the acid tolerance of the roots of sugarcane cultivar NiF8 and E. arundinaceus accession JW630 under various treatments of acid solutions in laboratory. Additionally, cellular damage of root was observed anatomically and their growth in acidic soil was verified by pot cultivation. To detect the interspecific difference in injury index of root by measuring electrolyte leakage, the following method was found suitable: initial washing for 15 min, acid stress treatment with 3% Al 2 (SO 4 ) 3 solution for 2 h, and leaking electrolyte from the damaged cells in ultrapure water for 2-4 h. The index was aligned with the relative growth rate (RGR) under acidic soil (pH 5.0), as E. arundinaceus with a lower index value exhibited a higher RGR. Callose, indicative of cellular damage, was deposited both after exposure to short-term and long-term acid stress in the exodermis of sugarcane, which showed higher injury index and lower RGR than those of E. arundinaceus, whereas callose deposition was not observed in E. arundinaceus even after acid stress treatments. Our results strongly suggest that the injury index measurements can be used to quantitatively evaluate differences in acid tolerance between sugarcane and Erianthus roots.