To understand the molecular basis of environment-induced and genotype-dependent chalkiness, six rice genotypes showing variable chalk levels were subjected to gene expression analysis during reproductive stages. In the high chalk genotypes, the peak expressions of ADP-Glucose Pyrophosphorylase (AGPase) Large Subunit 4 (AGPL4) occurred in the stages before grain filling commenced, creating a temporal gap with the upregulation of Granule Bound Starch Synthase I (GBSSI) and Starch Synthase IIA (SSIIA). Whereas, in the low chalk genotypes, AGPL4 expression generally occurred in later stages, close to the upregulation of GBSSI and SSIIA. However, heat treatment altered the expression pattern and created a gap between the expression peaks of AGPL4, and GBSS1 and SSIIA. This change was accompanied by transformed granular morphology, increased protein content, and chalkiness in the grains. AGPL4 expression pattern may partially explain chalkiness as it contributes to the pool of ADP-Glucose for producing amylose and amylopectin, the major components of the starch. Down-regulation of AGPase during grain filling stages could result in a limited pool of ADP-Glucose leading to inefficient grain filling and air pockets that contribute to chalkiness. The study suggests a mechanism of grain chalkiness based on the coordination of the three starch biosynthesis genes in rice.