The risk of acquiring variant Creutzfeldt–Jakob disease is closely related to polymorphism at codon 129 of the human prion gene, because almost all variant Creutzfeldt–Jakob disease patients are Met/Met homozygotes. Although animal transmission experiments corroborated this seeding discrimination, the origin of the differential seeding efficiency of the bovine prion seed for human codon 129 polymorphism remained elusive. Here, we used a short prion protein (PrP) peptide as a model system to test whether seeding discrimination can be found in this simple system. We used a previously developed ‘seed‐titration method’ and time‐resolved CD spectroscopy to compare sequence‐dependent seeding efficiency regarding codon 129 polymorphism. Our results showed that the Met→Val substitution on the human PrP (huPrP) peptide decreased seeding efficiency by 10 times when fibrils formed from bovine PrP (bPrP) peptide were used as the seed. To explore whether the different seeding barrier is due to the chemical and structural properties of Met and Val or whether another residue is involved in this peptide model, we constructed three bPrP mutants, V112M, L138I and N143S, in each of which one residue was replaced by the corresponding human residue. Our data showed that Leu138 in the bPrP seed might be the key residue causing the different seeding efficiencies related to 129M/V polymorphism and the interference effect of huPrP129V in the huPrP129M/V mixture. We propose a ‘surface competition hypothesis’ to explain the big seeding barrier caused by 129V in the PrP peptide seeding experiment.Structured digital abstract  huPrP aggregates with bPrP by circular dichroism (View Interaction 1, 2)  bPrP aggregates with bPrP by circular dichroism (View interaction)  bPrP aggregates with bPrP by electron microscopy (View interaction)  bPrP aggregates with bPrP by fluorescence technology (View interaction)  huPrP aggregates with huPrP by electron microscopy (View interaction)  huPrP aggregates with huPrP by fluorescence technology (View interaction)  huPrP aggregates with huPrP by circular dichroism (View interaction)