Global wheat production is estimated to fall by 6% for each 1oC increase in temperature. However, the effect of nighttime temperature (Tmin) on grain yield and genetic loci associated with them is not known. Since 70% of the global wheat production comes from irrigated or high rainfall regions, our focus was to study the effect of Tmin in high yielding environments. To address this question, we grew a spring wheat association mapping panel (WAMI) of 287 lines in a wide range of environments in different climatic conditions. Our analysis indicated that Tmin at heading is more detrimental than the Tmin at vegetative and grain filling stages in spring wheat. Reaction norm models were used to investigate and dissect the genetic basis of the differential response of genotypes to nighttime temperature through changes in grain yield (GY) and thousand-grain weight (TGW) in response to change in Tmin. The effect of Tmin on GY and TGW was greater than the maximum average daytime temperature in our study. We plotted yield of each genotype in each environment against Tmin of the environment and slope of the response of each genotype was used for GWAS. We found 139 loci associated with Tmin for GY and 178 loci associated with Tmin TGW, among them 101 loci were common between them. The locus that was involved in the variation of trait per se (Vrn-A1 for heading; in chromosome 5A at 90 cM), TGW locus in chromosome 5A at 98cM, and in chromosome 6A at 79-85cM (TaGW2 gene) were involved in the response to Tmin. Our approach provides a model to dissect the genetic basis of the response of genotypes to nighttime temperature.