Oligopeptides from grape must represent a secondary source of nitrogen for yeasts to grow and carry out fermentation. Saccharomyces cerevisiae takes up oligopeptides from the environment through multiple oligopeptide transporters with different peptide length specificities. However, due to difficulties associated with the qualitative and quantitative measurement of peptides in natural matrices, peptide transporter specificities have been mostly researched in single peptide environments. Using a peptide mapping method, we monitored the relative consumption of peptides derived from a protein hydrolysate by a set of CRISPR-Cas9-engineered S. cerevisiae wine strains to study oligopeptide transporters from the Opt and Fot families. Results show that Opt2 can import peptides containing three to -at least- seven amino acid residues, which is a broader peptide length specificity than previously reported, while Opt1 was not functional as a peptide transporter in these strains. Fot1, Fot2 and Fot3, previously referred to as di-tripeptide transporters in S. cerevisiae wine strains, could also import tetrapeptides. The consumption order of peptides was determined by the peptide length as higher chain length peptides were taken up by Opt2 only after most di-tetrapeptides were depleted from the media. Altogether, Fot and Opt2 activity assured completion of the fermentation process without necessarily requiring ammonia or free amino acids. Analysis of peptide transporter gene expression during fermentation showed an effect of SO42- not only on Opt1, but also on Fot, and supported the assumption of a possible interplay between Fot and Opt2 activities.