Laboratory experiments were conducted at the Department of Crop Physiology, Tamil Nadu Agricultural University, Coimbatore during 2019-2020 by using Temperature Induction Response (TIR) technique. The popular cotton (Gossypium hirsutum L.) varieties of Tamil Nadu like KC 3, SVPR 2 and MCU 7 were taken for the screening of high temperature tolerance. The identical sized five days and seven days old cotton seedlings were selected and exposed to inductive temperature (gradual temperature raised from 28 to 40℃) for 4 h and non-inductive temperature (46℃ for 3 h, 47℃ for 3 h, 48℃ for 3 h and 48℃ for 4 h) for specific time duration. Among the genotypes, KC 3 had recorded the highest thermotolerance in terms of seedling survival, cell viability, total soluble protein and lipid peroxidation (MDA content) as compared to other two varieties under both inductive and non-inductive temperature.