Heat stress adversely affects wheat production in many regions of the world and is particularly detrimental during reproductive development. The objective of this study was to identify quantitative trait loci (QTL) associated with improved heat tolerance in hexaploid bread wheat (Triticum aestivum). To accomplish this objective, an analysis of both the phenotypic and genetic responses of two recombinant inbred line (RIL) populations was conducted. RIL populations Halberd x Cutter and Halberd x Karl 92 (H/K) both derive heat tolerance from Halberd and segregate in their response to heat stress. A heat susceptibility index (HSI) was calculated from the reduction of three yield components; kernel number, kernel weight, and single kernel weight, following a three-day 38 degrees C heat stress treatment during early grain-filling. The HSI, as well as temperature depression of the main spike and flag leaf were used as measurements of heat tolerance. Genetic linkage maps were constructed for both populations and were used in combination with phenotypic data and statistical software to detect QTL for heat tolerance. In a comparison across the two across populations, seven common QTL regions were identified for HSI, located on chromosomes 1B, 3B, 4A, 5A, 5B, and 6D. Subsequent analysis of temperature depression in the H/K population identified seven QTL that co-localized for both cooler organ temperature and improved HSI. Four of the beneficial alleles at these loci were contributed Halberd. The genetic effect of combining QTL, including QHkw.tam-1B, QHkwm.tam-5A.1, and QHskm.tam-6D showed the potential benefit of selection for multiple heat tolerant alleles simultaneously. Analysis of the H/K population in the field under abiotic stress detected QTL on chromosome 3B and 5A, which were in agreement with results from the greenhouse study. The locus QYld.tam-3B was pleiotropic for both temperature depression and HSI in both experiments and was associated with higher biomass and yield under field conditions. The results presented here represent a comprehensive analysis of both the phenotypic response of wheat to high temperature stress and the genetic loci associated with improved heat tolerance and will be valuable for future understanding and improvement of heat stress tolerance in wheat.