Starch is a natural stored carbohydrate resource for the human diet, synthesized and stored in amyloplasts in higher plants. Starch synthase III is directly linked to the amylopectin chain’s elongation in wheat grains, but global warming is a critical barrier to its activity. The present investigation includes the characterization of the TaSS-IIIa1D gene in two wheat genotypes, viz. PBW-343 (heat susceptible) and IC252874 (heat tolerant). The genomic DNA of the above genotypes was used for PCR amplification of TaSS-IIIa1D gene using overlapping gene-specific primers and cloning the gene using the pJET1.2/blunt cloning vector, followed by sequencing and detection of SNPs. In-silico analysis revealed that the TaSS-III gene contains two homologs, TaSS-IIIa and TaSS-IIIb, which were found on the plus strand of chromosome 1 (1A, 1B and 1D) and minus strand of chromosome 2 (2A, 2B and 2D), respectively. The genomic size of the TaSS-IIIa1D gene was approximately 10,529 bp. All homeologous copies of the gene contained 16 exons. Twenty-nine specific SNPs were identified in PBW343 and twenty in IC252874 genotypes for the gene vis-a-vis with the reference copy of Chinese spring cultivar. There were 14 intronic and 15 exonic SNPs contributing to 18 transitions and 9 transversions in the genotype PWB-343, reflecting the transition bias, while in genotype IC252874, nine transitions, nine transversions, and two deletions contribute to 6 intronic and 14 exonic SNPs showing no such bias. Eighteen SNPs detected between the two genotypes for the gene consisting of 11 transitions and 7 transversions also showed transition bias. Third exon found most variable with the maximum number of SNPs. Seven SNPs were found associated with SBD-1, SBD-2, and SS-CD domains of the TaSS-IIIa1D protein. SNPs identified between the heat-sensitive and heat-tolerant genotypes for the heat-sensitive enzyme coding gene could be utilized for wheat improvement after proper validation.