Fungal Oligopeptide Transporters (Fot) have been described as a family of di- and tripeptide transporters in fungi. In Saccharomyces cerevisiae, Fot are only present in wine strains as a result of a horizontal gene transfer (HGT) from the yeast Torulaspora microellipsoides. The S. cerevisiae wine strain 59A, haploid derivative of the EC1118 strain which contains Fot1 and Fot2, displays a better fermentation performance and higher cell viability by the end of fermentation than its Fot-knockout counterpart; additionally, the higher capacity to consume oligopeptides due to Fot is also responsible for a more positive aroma profile of the strain 59A during wine fermentation, with a lower production of acetate and higher production of ester acetates and fusel alcohols. These results evidenced the adaptive advantage conferred by Fot to S. cerevisiae within the wine environment, also highlighting the interest in using Fot-containing strains for winemaking. Despite the benefits associated with Fot, little is still known about this family of transporters. Recently, we have reported that Fot members in S. cerevisiae and T. microellipsoides have different oligopeptide specificities despite their high sequence identity. To explain these differences, we have combined AlphaFold modelling and molecular docking with selected oligopeptides as ligands to propose a mechanism for substrate binding. Our findings point at highly conserved residues located at the loose segments in transmembrane domains 1 and 6 as to shape the binding site of Fot, suggesting that variable sites among Fot members, localized in the N-terminal domains and inter-transmembrane regions, may be responsible for their distinct oligopeptide uptake abilities. We have studied the conservation of the candidate residues for substrate binding in a dataset of putative Fot sequences identified through a curated strategy. With a limited number of sequences found in Basidiomycota yeast species, homologous FOT gene sequences were mainly identified in Ascomycete yeasts and filamentous fungal species from the Eurotiales order. Phylogenetic reconstruction of putative Fot members in Ascomycota reveals a patchy distribution that indicates more potential cases of HGT or gene duplications