Although cotton is sun loving crop but its production is adversely affected due to biotic and abiotic stress, heat stress contributed significantly in this loss around the globe. The present research work on heat tolerance in cotton is a step forward to understand the genetic basis of heat tolerance in upland cotton. The presence and identification of genetic variation for certain traits is one of the pre-requisite to start research work. There are several techniques for the screening of germplasm for heat tolerance. Relative cell injury was used for the identification of the most heat tolerant and susceptible cotton genotypes i.e. VH-259, VH-142 and DNH-40, VH-282 respectively. The identified genotypes were used for the development of F1, F2, BC1 and BC2 populations for genetic studies. The biometrical analysis revealed that selected traits i.e. relative cell injury, chlorophyll contents, boll shedding percentage, plant height, number of bolls per plant, number of seeds per boll, seed cotton yield were controlled by additive and non-additive type of gene action including epistatic effects. It is suggested that selection in these populations should be done in later generations for the development of new germplasm with enhanced heat tolerance.