Although the relationship between basal (BT), acquired (AT) and maintenance of acquired thermotolerace (MT) has been illustrated in a heat resistant tomato genotype and sensitive tomato genotypes, whether more resistant and sensitive tomato genotypes are satisfied with the rule and the relationships of antioxidant enzymes (AE) activity in three kinds of thermotolerance are not known. Here, we have observed the intension of three kinds of tomato thermotolerance is associated with AE activities. The priming and enhancement of thermotolerance was temperature dependent with stronger thermotolerance priming by moderately high temperatures than warmer temperatures. Interestingly, AE activity also showed significantly higher in seedlings under moderately high temperature than ones at warmer temperatures. While after the first optimized priming of different high temperatures combined with secondary priming for a reasonable period, AE activity and its thermotolerance further enhanced. Surprisingly, these optimized acclimation treatments showed no difference in AE activity and MT intension, suggesting that secondary priming could supply gaps produced by the first priming of warmer high temperatures, through enhancing AE activity. Additionally, the basal heat resistant genotypes showed stronger AE activity and thermotolerance (AT and MT) than sensitive genotypes. Results from this study will provide insights into understanding mechanism behind regulating tomato thermotolerance and facilitate the development of heat tolerant cultivars.