Starch structure and reactivity is not fully understood, despite the widespread use of starch and its derivatives in food and non-food industries. This study is focused on the relationship between starch granular and molecular structure, and their impact on the relative extent of reaction (i.e., reactivity). Starch was derivatized with a fluorescent probe (5-[4,6-dichlorotriazinyl]aminofluorescein) as a derivatizing reagent (model derivatization system). The granular locale of reaction, and relative reactivities of debranched starch amylose (AM) and amylopectin (AP) branch chains [long (LC), medium (MC), and short (SC)] were monitored for 24 h via confocal laser scanning microscopy (CLSM) and size-exclusion chromatography (SEC) equipped with refractive index (RI) and fluorescence (FL) detection, respectively. The granular locale of reaction was initially focused at external granule surfaces (≤ 0.5 h), and gradually progressed into granule matrix during latter reaction stages (12-24 h). Starch chain reactivities decreased in the order: AP-LC >> AM, AM-MC > AP-SC, irrespective of the length of reaction. Later, in the modified SEC set up, ‘intermediate material’ was identified (IM, subpopulation of AM/AP-LC, 100 ≤ degree of polymerization <372), which exhibited 5.0-9.4 fold higher reactivity than the overall reactivity calculated across all starch chains. IM was shown to be a primary molecule in the outer layers of starch granules where the reaction was most intensive. Overall findings suggest that granule architecture impacts the relative reactivities and reaction patterns of AM and AP branch chains. The gathered information is expected to improve technical practice and design specific functionality of starch derivative.