Aims: The main objective of this study was to identify amino acid residues in the AGT1-encoded α-glucoside transporter (Agt1p) that are critical for efficient transport of maltotriose in the yeast Saccharomyces cerevisiae. Methods and Results: The sequences of two AGT1-encoded α-glucoside transporters with different efficiencies of maltotriose transport in two Saccharomyces strains (WH310 and WH314) were compared. The sequence variations and discrepancies between these two proteins (Agt1pWH310 and Agt1pWH314) were investigated for potential effects on the functionality and maltotriose transport efficiency of these two AGT1-encoded α-glucoside transporters. A 23-amino-acid C-terminal truncation proved not to be critical for maltotriose affinity. The identification of three amino acid differences, which potentially could have been instrumental in the transportation of maltotriose, were further investigated. Single mutations were created to restore the point mutations I505T, V549A and T557S one by one. The single site mutant V549A showed a decrease in maltotriose transport ability, and the I505T and T557S mutants showed complete reduction in maltotriose transport. Conclusions: The amino acids Thr505 and Ser557, which are respectively located in the transmembrane (TM) segment TM11 and on the intracellular segment after TM12 of the AGT1-encoded α-glucoside transporters, are critical for efficient transport of maltotriose in S. cerevisiae. Significance and Impact of the Study: Improved fermentation of starch and its dextrin products, such as maltotriose and maltose, would benefit the brewing and whisky industries. This study could facilitate the development of engineered maltotriose transporters adapted to starch-efficient fermentation systems, and offers prospects for the development of yeast strains with improved maltose and maltotriose uptake capabilities that, in turn, could increase the overall fermentation efficiencies in the beer and whisky industries.