Technical Abstract: The granule-bound starch synthase (GBSS) is the enzyme responsible for amylose synthesis in starch granules. Loss of GBSS activity results in starch granules containing mostly amylopectin and little or no amylose, a phenotype described as waxy. Previously, two phenotypic classes of waxy alleles were identified in sorghum (Sorghum bicolor L. Moench) characterized by the absence (waxya; wxa) or presence (waxyb; wxb) of the GBSS protein in the endosperm. To characterize these alleles, we examined endosperm architecture using Scanning Electron Microscopy (SEM), assayed GBSS enzymatic activities, and identified DNA lesions associated with the mutations in the GBSS (Sb10g002140) gene. wxa, the allele present in B Tx630 and R Tx2907 contained a large insertion in the third exon, which was consistent with the absence of the GBSS protein previously observed. wxb, the allele present in B 9307 and R TxAGR1 contained a missense mutation that resulted in conversion of glutamine 268 to histidine in a conserved domain in starch synthases. In wxb, GBSS activity was less than 20% that of the non-waxy line B Wheatland, and GBSS activity was not detected in wxa. The protein model of GBSS provided some insight into the function of the gene. SEM showed that endosperm architecture was very similar in both wxa and wxb alleles, but altered in comparison to non-waxy lines R Tx430 and B Wheatland. Both alleles may have a range of potential applications in grain sorghum, because of low amylose content in their starch and the presence or absence of the GBSS.