Thioflavin-T is one of the most important amyloid specific dyes and has been used for more than 50 years; however, the molecular mechanism of staining is still not understood. Chemically synthesized short polyglutamine peptides (Q_n, n = 5–10) were subjected to the thioflavin-T (ThT) staining assay. It was found that the minimum Q_n peptide that stained positive to ThT was Q₆. Two types of ThT-binding sites, a high-affinity site (k_d1 = 0.1–0.17 μM) and a low-affinity site (k_d2 = 5.7–7.4 μM), were observed in short polyQs (n = 6–9). ¹³C{²H}REDOR NMR experiments were carried out to extract the local structure of ThT binding sites in Q₈ peptide aggregates by observing the intermolecular dipolar coupling between [3-Me-d₃]ThT and natural abundance Q₈ or residue-specific [1,2-¹³C₂] labeled Q₈s. ¹³C{²H}REDOR difference spectra of the [3-Me-d₃]ThT/natural abundance Q₈ (1/9) complex indicated that all of the five carbons of the glutamine residue participated in the formation of ThT-binding sites. ¹³C{²H}DQF–REDOR experiments of [3-Me-d₃]ThT/residue-specific [1,2-¹³C₂] labeled Q₈ (1/50) complexes demonstrated that the N-terminal glutamine residue had direct contact with the ThT molecule at the high-affinity ThT-binding sites.