In Φ-value analysis, the effects of mutations on the folding kinetics are compared with the corresponding effects on thermodynamic stability to investigate the structure of the protein-folding transition state (TS). Here, molecular dynamics (MD) simulations (totaling 0.65 ms) have been performed for a large set of single-point mutants of a 20-residue three-stranded antiparallel β-sheet peptide. Between 57 and 120 folding events were sampled at near equilibrium for each mutant, allowing for accurate estimates of folding/unfolding rates and stability changes. The Φ values calculated from folding and unfolding rates extracted from the MD trajectories are reliable if the stability loss upon mutation is larger than approximately 0.6 kcal/mol, which is observed for 8 of the 32 single-point mutants. The same heterogeneity of the TS of the wild type was found in the mutated peptides, showing two possible pathways for folding. Single-point mutations can induce significant TS shifts not always detected by Φ-value analysis. Specific nonnative interactions at the TS were observed in most of the peptides studied here. The interpretation of Φ values based on the ratio of atomic contacts at the TS over the native state, which has been used in the past in MD and Monte Carlo simulations, is in agreement with the TS structures of wild-type peptide. However, Φ values tend to overestimate the nativeness of the TS ensemble, when interpreted neglecting the nonnative interactions.