AbstractStarch biosynthesis is a complex process underlying grain chalkiness in rice in a genotype‐dependent manner. Coordinated expression of starch biosynthesis genes is important for producing translucent rice grains, while disruption in this process leads to opaque or chalky grains. To better understand the dynamics of starch biosynthesis genes in grain chalkiness, six rice genotypes showing variable chalk levels were subjected to gene expression analysis during reproductive stages. In the chalky genotypes, peak expression of the large subunit genes ofADP‐glucosepyrophosphorylase(AGPase), encoding the first key step in starch biosynthesis, occurred in the stages before grain filling commenced, creating a gap with the upregulation of starch synthase genes,granule bound starch synthase I(GBSSI) andstarch synthase IIA(SSIIA). Whereas, in low‐chalk genotypes,AGPaselarge subunit genes expressed at later stages, generally following the expression patterns ofGBSSIandSSIIA. However, heat treatment altered the expression in a genotype‐dependent manner that was accompanied by transformed grain morphology and increased chalkiness. The suppression ofAGPasesubunit genes during early grain filling stages was observed in the chalky genotypes or upon heat treatment, which could result in a limited pool of ADP‐Glucose for synthesizing amylose and amylopectin, the major components of the starch. This suboptimal starch biosynthesis process could subsequently lead to inefficient grain filling and air pockets that contribute to chalkiness. In summary, this study suggests a mechanism of grain chalkiness based on the expression patterns of the starch biosynthesis genes in rice.