Background and objectives:The role of amylolytic activities during four stages of pregermination varying on embryonic growth length between 0.5 and 7.0 mm on dynamic changes of rice kernel morphology and chemical composition, crystallinity, and thermal properties of isolated starches from three different Thai rice cultivars-SPT1 (waxy), PTT1 (low amylose), and PNL2 (high amylose)-were investigated. Findings: Amylolytic activities of all rice cultivars significantly increased at each progressive stage, whereas SPT1 showed the highest increase in alpha-amylase activity as the first stage of pregermination. Scanning electron micrographs showed the radial crack of kernel, appearance of individual and nonuniform starch granules after pregermination, particularly of the SPT1 cultivar. Molecular weight distribution of pregerminated brown-rice starch (PGBRS) indicated the partial hydrolysis of starch molecules especially amylopectin, resulting in a decrease fraction of high MW molecules after pregermination. All PGBRS had A-type X-ray diffraction patterns with higher relative crystallinity than their natives, except that of PNL2 cultivar. Pregermination led to the change onset temperature (To) of starch gelatinization (gel_p1) and retrograded amylopectin melting (ret_p1) of PGBRS for all rice cultivars and peak temperature (Tp) of gel_p1 and ret_p1 of PGBRS for only SPT1 cultivar, whereas SPT1 was found at the lower stage than other cultivars. PGBRS at the second and third stage for SPT1, after the third stage for PTT1, and after the second stage for PNL2 had lower enthalpy (DH) of ret_p1 than their natives. Conclusions: Alpha-amylase activity plays a role in partial hydrolysis of amylopectin at amorphous region during rice pregermination which resulted in significant changes of thermal properties for all rice, especially SPT1 cultivar. Pregermination with 2-3-mm EGL of all rice could be used as starch bio-modification to obtain a lower retrograded starch as a functional ingredient of rice-based products. Significance and novelty: The change in thermal properties of waxy SPT1 rice cultivar, which mostly contained amylopectin and highly increased alpha-amylase activity after first stage of pregermination, was more pronounced than those of nonwaxy PTT1 and PNL2 rice cultivars. Cereal Chemistry. 2018;95:543-554. wileyonlinelibrary.com/journal/cche © 2018 AACC International | 543 K E Y W O R D S amylolytic activities, isolated rice starch, kernel morphology, physicochemical properties, pregermination How to cite this article: Kupkanchanakul W, Thongngam M, Shi Y-C, Naivikul O. Role of amylolytic activities during pregermination on rice kernel morphology and physicochemical properties of isolated starch. Cereal Chem. 2018;95:543-554.