The glycemic index (GI) of rice is a complex trait, affected by amylose content (AC), size, and packaging of starch granules (SGs). In this study, the electron microscopy results of starch morphology of nine rice genotypes showed varying AC (6.93–36.9%), and the predicted GI (pGI: 41.07–82.46) in relation to genetic factors revealed that smaller SG surface area (20.06 µm²) and irregular morphology (Hap 3-3 P-11, pGI = 41.07) were associated with a lower pGI, while larger SG surface area (47.68 µm²) and spherical structure were associated with a higher pGI (NON-HAI, pGI = 82.46). The expression of starch biosynthesis and packaging-related genes (OsSSIIb, OsSSIIc, OsSBEIIa, OsISA1, OsISA3, OsGBP, OsFLO6, and OsBT1) revealed downregulation of OsGBP and OsISA3 genes in low pGI lines IRRI-147 (pGI = 56.2) and Hap 3-1-p-18 (pGI = 41.79), respectively, while higher levels of expression of the OsBT1 gene in Makro (pGI = 59.06) and OsSSIIb in Swarna (pGI = 58.06) were observed. All these genotypes had similar AC (~30%), but the difference in expression pattern was correlated with starch granule morphology, suggesting its role in influencing pGI. Further, analysis of allelic variation in eight starch-related genes across 20 rice genotypes showed that allelic variants of only OsGBP were correlated with AC, where allele group 2 showed lower AC (9.62%), while all other allele groups showed consistently high AC (22–24%). These findings underscore the critical role of starch granule morphology and OsGBP allelic variation in determining AC and GI, providing actionable insights for developing low GI rice varieties using tools like CRISPR.