Rice lines with slower starch digestibility provide opportunities in mitigating the global rise in type II diabetes and related non-communicable diseases. However, screening for low glycemic index (GI) in rice breeding programs is not possible due to time and cost constraints. This study evaluated the feasibility of using in vitro cooked grain amylolysis, starch mobilization patterns during seed germination, and variation in starch structure and composition in the mature seed to differentiate patterns of starch digestibility. Mobilization patterns of total starch, resistant starch, amylose and amylopectin chains, and free sugars during seed germination revealed that the process is analogous to digestion in the human gastrointestinal tract. The combination of these biochemical markers can be used as an alternative measure to predict GI. Additionally, transcriptome analysis of stored mRNA transcripts in high and low GI lines detected differences in starch metabolism and confirmed the importance of seed storage pathways in influencing digestibility. Pathway analyses supported by metabolomics data revealed that resistant starch, cell wall non-starch polysaccharides and flavonoids potentially contribute to slower digestibility. These new insights can guide precision breeding programs to produce low GI rice with acceptable cooking quality to help mitigate the burden of diet-associated lifestyle diseases.