Resistant starch (RS) has been proven to benefit human health as dietary fiber. High RS in wheat grains is positively correlated with amylose content, which helps prevent Type 2 diabetes, obesity, colon cancer and cardiovascular diseases. Altering amylose content can be achieved via genetic modification of starch composition by deactivating of starch branching enzyme IIa (SBEIIa), a key player in amylopectin biosynthesis pathway, resulting in increased amylose proportion. In our breeding practice, high amylose wheat lines were produced using a SBEIIa homeolog triple-null mutant as one parent followed by backcrosses and marker-assisted selection (MAS). Kompetitive Allele-Specific PCR (KASP) markers specific for each sbeiia gene in hexaploid wheat were designed and validated to screen large F2 and/or backcross populations. These KASP markers allowed quick and accurate selection of individuals with desirable agronomical traits while increased amylose and RS contents up to 55% and 11%, respectively. Quantitative real-time PCR (qRT-PCR) was performed to analyze transcripts for the target homeologous genes over a time course during grain development in an F6 line. Results showed that expression of the SBEIIa genes in the F6 line behaved similar to that of its mutant parent. Starch granule morphology from the modified advanced lines were analyzed with scanning and transmission electron microscopy. The rheology and processing quality of wholemeal flour was assessed using Rapid Vsico Analyzer (RVA) and some end-use products. To summarize, it is demonstrated that these KASP markers can effectively yet precisely assist breeding for high content of amylose and RS wheat in a high throughput manner.