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Reserve starch of cereal crop accounts for about 70％ of grain endosperm and acts as an important human carbohydrate resource worldwide. Wheat reserve starch is synthesized by enzymatic machinery in endosperm cells. To identify genes involved in starch biosynthesis, we constructed 30 RNA-Seq libraries of 10 endosperm-development periods and performed expression and localization analyses. Of 166 endosperm-expressed homologs of starch biosynthesis-related genes, 74 showed expression correlated with reserve starch accumulation, including 26 with expected subcellular distribution and higher expression than their isoforms. The key proteins SUS3, UGP1, cAGPase, and Bt1-3 formed the main metabolic pathway and contributed the major substrates for starch processing in amyloplasts. Important isoforms, key pathway proteins, and the main carbon flux toward starch formation in the reserve starch biosynthesis pathway were identified. Based on a co-expression analysis, a library of 425 transcription factors was produced to screen for common regulators. TaMYB44 had features of transcription factors and bound to TaSUT1, TaSSIIIa, TaBEIIa, TaISA1, and TaBEIIb promoters in yeast, suggesting that the gene is a pathway regulator. This study sheds light on understanding the mechanism of reserve starch biosynthesis and will be helpful for increasing starch content in wheat endosperm via biotechnological strategies.