The objective of present study was to examine the presence of variation in relative cell injury percentage (RCI%), as a measure of heat tolerance in sample of G. hirsutum germplasm and also to investigate genetic basis, if any, of RCI% under heat stress. For this purpose, response of 70 cotton varieties/lines was studies to optimum and high temperature, and RCI% was measured at reproductive stage. Data showed differing responses of the germplasm to the two temperature regimes. On the basis of similarities MNH 552, FH 100 and NIAB 111 were screened out as tolerant whilst Cedix ST-362 (GL), LRA 5166 and 4F were identified as susceptible varieties. Three sets of crosses involving tolerant × susceptible varieties were made according to generation mean technique. Six generations of each cross was field planted under optimum and high temperature and at reproductive stage RCI% was again measured. Genetic analysis of the data showed the predominant effect of additive component in the inheritance of RCI% under both the temperature regimes and consequently estimates of h2 ns were high in three crosses. These estimates were used to calculate response to selection in F3 population that appear to be encouraging. The results of present investigation suggest that RCI% could be used as effective selection criteria for selecting the plants with lower value of RCI%.