Amylose synthesis is strictly associated with activity of granule-bound starch synthase (GBSS) enzymes. Among several crops there are cultivars containing starch types with either little or no amylose known asnear-waxyorwaxy. This (near) amylose-free phenotype is associated with a single locus (waxy) which has been mapped to GBSS-type genes in different crops. Mostwaxyvarieties are a result of either low or no expression of a GBSS gene. However, there are somewaxycultivars where the GBSS enzymes are expressed normally. For these types, single nucleotide polymorphisms have been hypothesized to represent amino-acid substitutions leading to loss of catalytic activity. We here confirm that the HvGBSSIa enzyme from one suchwaxybarley variety, CDC_Alamo, has a 90% reduction in catalytic activity. We also engineered plants with expression of transgenic C-terminal green fluorescent protein-tagged HvGBSSIa of both thenon-waxytype and of the CDC_Alamo type to monitor their subcellular localization patterns in grain endosperm. HvGBSSIa fromnon-waxycultivars was found to localize in discrete concentric spheres strictly within starch granules. In contrast, HvGBSSIa fromwaxyCDC_Alamo showed deficient starch targeting mostly into unknown subcellular bodies of 0.5-3 μm in size, indicating that the waxy phenotype of CDC_Alamo is associated with deficient targeting of HvGBSSIa into starch granules.