Starch is one of the major components of cereals and many fruits. Granule-bound starch synthase (GBSS) genes, responsible for amylose synthesis, have been used to study phylogenetic and evolutionary relationships in many plant species due to their low copy number. In this study, sequences of GBSS genes isolated from three fruit trees, including apple, peach, and orange, together with an additional 22 GBSS sequences from various monocots and eudicots, available in public databases, were used to investigate a comprehensive evolutionary history of the GBSS gene across monocots and eudicots. It is revealed that the structure of GBSS genes in monocots and eudicots is conserved and all sequences must have evolved from a common ancestor. Moreover, the GBSS gene in the ancestral angiosperm must have undergone a duplication event approximately 251 million years ago (MYA) to generate two families: GBSSI and GBSSII. Both GBSSI and GBSSII are present in monocots; whereas, GBSSI is absent in eudicots. Thus, a major divergence event in the ancestral GBSSII, ~165 MYA when monocots and dicots split, has given rise to monocot-specific GBSSII and eudicot GBSS genes. In eudicots, multiple GBSS genes within the same species have high levels of sequence divergence although they have higher identity among each other than with either GBSSI or GBSSII genes from monocots. Overall these findings suggest that GBSSII of monocots must have an orthologous relationship with GBSS genes of eudicots. As in monocots, GBSSI and GBSSII are expressed in both storage and non-storage tissues, respectively, and they may be associated with neofunctionalization of starch synthase.