Lack of nutritionally appropriate foods is one of the leading causes of obesity in the US and worldwide. Wheat (Triticum aestivum) provides 20% of the calories consumed daily across the globe. The nutrients in the wheat grain come primarily from the starch composed of amylose and amylopectin. Resistant starch content, which is known to have significant human health benefits, can be increased by modifying starch synthesis pathways. Starch synthase enzyme SSIIa, also known as starch granule protein isoform-1 (SGP-1), is integral to the biosynthesis of the branched and readily digestible glucose polymer amylopectin. The goal of this work was to develop a triple null mutant genotype for SSIIa locus in the elite hard red winter wheat variety ‘Jagger’ and evaluate the effect of the knock-out mutations on resistant starch content in grains with respect to wild type. Knock-out mutations in SSIIa in the three genomes of wheat variety ‘Jagger’ were identified using TILLING. Subsequently, these loss-of function mutations on A, B, and D genomes were combined by crossing to generate a triple knockout mutant genotype Jag-ssiia-∆ABD. The Jag-ssiia-∆ABD had an amylose content of 35.70% compared to 31.15% in Jagger, leading to ~ 118% increase in resistant starch in the Jag-ssiia-∆ABD genotype of Jagger wheat. The single individual genome mutations also had various effects on starch composition. Our full null Jag-ssiia-∆ABD mutant showed a significant increase in RS without the shriveled grain phenotype seen in other ssiia knockouts in elite wheat cultivars. Moreover, this study shows the potential for developing nutritionally improved foods in a non-GM approach. Since all the mutants have been developed in an elite wheat cultivar, their adoption in production and supply will be feasible in future.