<p>Starch is the main storage carbohydrate in plants and serves as a vital resource for human consumption and various industries. Granule-bound starch synthase (GBSS) is the key enzyme in starch synthesis, yet the evolution of its structural diversity in flowering plants is not well understood. This study aims to answer: what is the evolutionary history of the gene encoding the GBSS enzyme in angiosperms, and how does the structural diversity among its isoforms manifest? In addition, what are the key domains and residues that influence enzymatic function? We conducted a comprehensive phylogenetic analysis of GBSS-encoding genes to elucidate its evolutionary history and employed homology modeling to determine the enzyme’s structure by identifying important protein domains and motifs. Molecular dynamics (MD) simulations were performed to assess protein stability, considering mutations in specific residues. Our results indicate that GBSS is highly conserved in angiosperms, with phylogenetic analysis revealing the evolution of two isoforms, GBSSI and GBSSII. This diversification may be linked to duplication events in plant genomes. Although the isoforms share similarities, they exhibit structural variances and amino acid substitutions. Additionally, destabilizing mutations in key residues within binding sites and conserved domains were influenced by purifying selection, which helps maintain protein stability. This study enhances our understanding of the evolutionary and structural history of the main enzymes involved in starch synthesis, supporting future research on starch biosynthesis and the evolutionary divergence of GBSS in plants. We emphasize the importance of considering isoforms when using GBSS to reconstruct phylogenetic trees.</p> Graphical Abstract <p></p>