Heat stress is a critical factor affecting global wheat production and productivity. In this study, out of 500 studied accessions a diverse panel of 126 wheat genotypes grown under twelve distinct environmental conditions was analyzed. Using 35K single-nucleotide polymorphism (SNP) genotyping assays and trait data on five biochemical parameters, including grain protein content (GPC), grain amylose content (GAC), grain total soluble sugars (TSS), grain iron (Fe), and zinc (Zn) content, six multi-locus GWAS models were employed for association analysis. This revealed 67 significantly associated QTNs linked to grain quality parameters, explaining phenotypic variations ranging from 3–44% under heat stress conditions. By considering the results in consensus to at least three GWAS models and three locations, the final QTNs were reduced to 17, with 14 being novel findings. Notably, two novel markers, AX-94461119 (chromosome 6A) and AX-95220192 (chromosome 7D), associated with grain iron and zinc, respectively, were validated through KASP approach. Candidate genes, such as chaperonin Cpn60/GroEL/TCP-1 family, P-loop containing nucleoside triphosphate hydrolases (NTPases), Bowman-Birk type proteinase inhibitor (BBI), and NPSN13 protein, were identified from the associated genomic regions, which could be potentially targeted for improving quality traits and heat tolerance in wheat.