Cotton is cultivated in a wide range of environments from tropical to subtropical regions. In India, approximately 70%of the total land area covered by cotton is grown under rain-fed conditions where cotton frequently experiences drought stress along with other stresses, e.g.,high temperature and high salinity. There is a need to develop a technique to screen a large number of genotypes for high temperature tolerance. In this study, a screening protocol was developed based on the principle of "acquired tolerance" in which exposure of seedlings to a sublethal level of specific stress is used to induce tolerance to a subsequent lethal level of stress. After adapting this temperature induction response (TIR) technique to cotton, several species and varieties were screened for thermotolerance. Among the tested entries, Old World cotton species showed better thermotolerance than New World cotton species. Among 36 diverse Gossypium hirsutum germplasm lines, significant variation in acquired thermotolerance was seen. Thermotolerant genotype G. hirsutum (H-28), identified by the TIR technique, demonstrated increased cell viability and protein synthesis capacity during alleviation from high temperature stress. Results suggested that TIR is a robust and powerful technique and can be used to screen breeding lines or germplasms to identify thermotolerant lines.