<p>Sorghum [<i>Sorghum bicolor</i> (L.) Moench] is a staple food for millions of people in Africa and South Asia. It is mainly consumed for its starch. The starch composition and structure in the seed endosperm determines cooking properties, processing quality, and starch digestibility. </p> <p>An assay to measure the alkali spreading value (ASV) of sorghum is described. The assay was used to identify sorghum EMS mutants with variation in starch composition. The ASV mutants (ASV+) exhibited a range of starch thermal properties with starch gelatinization temperatures (GT) being lower or higher than samples from Tx623 or Sepon82. The ASV+ phenotypes were found to be correlated with starch related traits such as enthalpy (r = −0.53) and range of starch GT (T_c-T_o) (r = 0.60). </p> <p>Genes controling the ASV phenotype of sorghum and their impact on starch quality traits are described. Whole genome re-sequencing of sorghum EMS mutants exhibiting an ASV+ phenotype was used to identify single nucleotide polymorphisms (SNPs) in candidate genes <i>Sobic.004G163700</i> and <i>Sobic.010G093400</i>. The two genes were identified as a <i>SbeIIb</i>, a putative sorghum homolog of <i>amylose extender,</i> and as a <i>SSIIa</i>, respectively. Linkage analysis showed that the mutations in <i>Sobic.010G093400</i> and <i>Sobic.004G163700</i> co-segregated with the ASV phenotype. The <i>ssIIa</i>-mutants exhibited normal amylose values, lower starch GT and lower final viscosity than the wild type. The <i>sbeIIb</i>-mutants exhibited higher amylose content, higher starch GT and lower peak and final viscosity with poor gel consistency compared to the wild type and <i>ssIIa</i>-mutants. An allele dosage test indicated that the <i>sbeIIb</i>-mutants had an allele dosage dependent effect on amylose content. Double mutants of <i>sbeIIb</i> and <i>ssIIa</i> showed that amylose content, starch thermal properties and paste viscosity profiles resemble the <i>sbeIIb</i> parent. </p> <p>A study of ASV phenotypes in a panel of more than 750 sorghum conversion lines revealed genetic variation for the ASV phenotype. A few SC-lines exhibiting stable expression of the ASV+ phenotype over two growing seasons. Most of these lines were described as belonging to the working group Nandyal, durra types from India described as producing ‘glutinous grains’. Whole genome resequencing discovered common SNPs in genes associated with starch biosynthesis. A genome wide association study (GWAS) identified a significant SNP that could be associated with the starch biosynthesis gene <i>Sobic.010G273800</i>, and with candidate genes <i>Sobic.010G274800</i> and <i>Sobic.010G275001</i> both annotated as glucosyltransferases. Grain samples from SC489, SC491, SC587 and SC589 exhibited a consistent ASV+ phenotype with lower or similar starch GT, similar amylose content, and similar high viscosity and gel consistency compared to controls.</p> <p> </p>