Gastrointestinal (GI) α‐synuclein (aSyn) detection as a potential biomarker of Parkinson's disease (PD) is challenged by conflicting results of recent studies. To increase sensitivity and specificity, we applied three techniques to detect different conformations of aSyn in GI biopsies obtained from a longitudinal, clinically well‐characterized cohort of PD patients and healthy controls (HC). A total of 163 tissue blocks were collected from 51 PD patients (113 blocks) and 21 HC (50 blocks).