Thermotolerance limit in aquatic organism is set by the ability to sustain aerobic scope to sudden temperature shifts. This study tested the genetic and plastic differences in thermotolerance of Daphnia that can be explained by the differences in the ability to retain mitochondrial integrity at high temperatures. Five genotypes with different biogeographic origins were acclimated to 18ᵒC and 25ᵒC. We developed a rhodamine 123 in-vivo assay to measure mitochondrial membrane potential and observed higher fluorescent in heat damaged tissues as the disruption of the mitochondrial membrane potential. Significant effects on temperature tolerance were observed with CCCP and DNP but not with NaN3. Effects of toxins were significant in temperature sensitive genotype and high concentration of lactate was observed in 18ᵒC acclimated genotype only. We conclude that genetic and physiological differences are intricately linked to the ability of sustaining aerobic respiration at high temperatures which sets limit to the thermotolerance.