Impact of elevated temperature on physiological and biochemical changes were evaluated in 5 commercial sugarcane genotypes and 2 wild species clones at two different growth phases. The study revealed that heat stress decreased chlorophyll content, chlorophyll stability index (CSI), SPAD value, maximum quantum efficiency of PSII photochemistry (F_v/F_m ratio), leaf gas exchange parameters, relative water content (RWC), and activities of nitrate reductase (NR), sucrose-metabolizing enzymes (SPS, SS, AI, NI) in all the genotypes and species clones. In contrast, elevated temperature induced an increase in proline, total phenolics content (TP), antioxidant enzyme activities (SOD and POX), lipid peroxidation (LP), membrane injury index (MII) and soluble sugar content in all clones. Principal component analysis based on physiological heat tolerance indexes could clearly distinguish sugarcane genotypes into three heat tolerance clusters. Noteworthy in comparison to the heat-sensitive varieties, sugarcane genotype that possessed higher degrees of heat tolerance Co 99004 displayed higher chlorophyll content, CSI, antioxidant enzyme activities, NR activity, RWC, total phenols, sucrose-metabolizing enzymes, soluble sugar content and leaf gas exchange and lower level of lipid peroxidation and membrane injury index.