[Objective]High temperature is one of the main influencing factors restricting the growth and development of tomato and the quality and yield of tomato. The study of the effect of exogenous myo-inositol on the physiological and biochemical indexes of tomato seedlings and its molecular mechanism of tolerance to high temperature stress provides a theoretical basis for the improvement of the heat resistance of tomato.[Methods]Tomato variety "NRP20-7" was used as the test material, water and myo-inositol treatments were sprayed separately at common temperature and high temperature.respectively, and the phenotypic changes of each treatment were observed and analyzed by hyperspectral imaging, and the physiological indexes of each treatment group were determined and analyzed the expression of heat stress responsive genes, antioxidant and ABA-related genes by qRT-PCR.. [Results] The 20 mmol/L myo-inositol treatment could effectively alleviate the damage of high temperature stress on tomato seedlings, reduce the heat damage index of tomato leaves, increase the maximum photochemical efficiency (Fv/Fm), and significantly increase the content of photosynthetic pigments.Exogenous myo-inositol significantly increased proline, soluble sugar, and peroxidase activity, and significantly decreased malondialdehyde, hydrogen peroxide, and superoxide anion contents in tomato seedlings at high temperature.Exogenous myo-inositol increased heat stress response genes, antioxidant-related genes, ascorbic acid synthesis genes, and decreased heat shock protein genes.In addition, exogenous myo-inositol is involved in the response to high temperature stress by regulating abscisic acid synthesis and signaling pathways affecting the level content of ABA in tomato seedlings. [Conclusion]Exogenous myo-inositol enhances tomato tolerance to high temperature stress by regulating complex biological processes in plants.