Different grain positions on the rice panicle have asynchronous flowering phenomenon. The difference between superior and inferior grains not only affects the yield, but also restricts the quality of rice. In this study, the cytokinin‐related transgenic lines VIN3‐LIKE 2 (OsVIL2)‐OX (increased endogenous cytokinin content), Oryza sativa A‐type response regulator (OsRR1)‐OX, and Early heading date 1 (Ehd1)‐OX (B‐type response regulator) are used to analyze nutrient accumulation and starch physicochemical properties of superior and inferior grains from the physiological level. The research conclusions are as follows: The overexpression transgenic plants of OsVIL2, OsRR1, and Ehd1 result in an increase of average starch granule size and solubility, but decrease in apparent amylose content of superior and inferior grains, which further reduce the surface order of superior and inferior grains, and increase the relative crystallinity of superior grains. Increased cytokinin content possibly accelerates the accumulation of carbohydrates in superior and inferior grains by promoting the expression of OsRR1. In addition, Ehd1 might be the main gene that affects the production of inferior grains, and Ehd1 is also involved in the regulation of the surface order structure and crystallinity of starch from superior and inferior grains by cytokinin.