Breeding for heat tolerance is complicated by the polygenic nature of the trait and genetic engineering awaits delineation of the molecular mechanisms. To understand heat-tolerance mechanisms in cotton (Gossypum hirsutum L.), the world’s most important natural fiber crop, this study compared expression of selected heat-stress response genes between heat-tolerant (VH260 and MNH456) and heat-sensitive (ST213 and ST4288) cotton genotypes. Cotton orthologs of selected Arabidopsis heat-stress response genes included two heat-stress transcription factors, three heat-shock proteins, and the general stress response genes: ascorbate peroxidase and the calcium-dependent stress responder, ANNAT8. The seedlings and flowering plants of each genotype were exposed to heat (38°C) for 1 to 3 hours or days, respectively, followed by RNA isolation from the seedlings and ovaries of flowering plants for gene expression analysis by real-time quantitative polymerase chain reactions. All genes, except the heat-shock protein GHSP26, were found to be induced exclusively in the heat-tolerant lines. The genotype VH260, which reportedly is tolerant to extreme heat (45°C), was found to display much higher induction of these genes than MNH456, the heat-tolerance level of which has not been thoroughly studied. Further, VH260 showed high gene induction in both seedlings and ovaries, whereas MNH456 showed lower induction levels. Strong heat tolerance in VH260 most likely is based on early induction of multiple mechanisms coordinately functioning to defend the plants against membrane damage, protein denaturation, and oxidative stress, in addition to rapid sensing of heat stress to signal the defense processes at the flowering stage towards minimizing yield losses and increasing boll retention during heat stress.