Temperature of the earth is increasing day by day due to global warming. Though Upland cotton is sun loving plant but its production is affected adversely due to increase in temperature not only in Pakistan but also in the whole world. That is why research work related to heat tolerance in cotton is a step forward. Objective of this study was to develop understanding the genetic basis of heat tolerance in upland cotton. The presence and identification of genetic variation for certain traits (plant height, number of bolls per plant, boll weight, boll shedding percentage, gin turn out, fibre length, fibre strength, fibre fineness, number of seeds per boll, seed cotton yield, relative cell injury and chlorophyll contents) is one of the prerequisite to start research work. There are several techniques for the screening of germplasm for heat tolerance. Relative cell injury was used in the present study 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 F2, BC1 and BC2 populations for genetic studies of heat tolerance and other agronomic traits. The results revealed that all the traits were controlled by both additive and non-additive type of gene action including epistatic effects. Genetic advance studies showed that F2 population could be used for selection in order to improve plant height, fibre strength, seed cotton yield and relative cell injury percentage. Hybrid breeding could be suggested in number of boll per plant, boll weight, boll shedding percentage, gin turn out, fibre length, fibre fineness, number of seeds per bolls and chlorophyll contents due to low genetic advance and high heritability. Transcriptomic analysis was also done in two heat tolerant genotypes VH-259 and VH-142 by using HSP related genes reported in the database from G. raimondii. The purpose of this experiment was to examine and identify the expression of these genes in G. hirsutum at variable temperature regimes. Certain HSP genes were identified in both genotypes in this experiment. Both these genotypes exhibited different pattern of heat stress tolerance based on differential expression of genes.