Background: Misfolded α‐synuclein in Parkinson's disease (PD) and dementia with Lewy bodies (DLB) can be detected using the real‐time quaking‐induced conversion (RT‐QuIC) technique in cerebrospinal fluid (CSF). Objectives: The objectives are (1) to examine misfolded CSF α‐synuclein incidence, and (2) to compare clinical presentation, sports history, brain volumes, and RT‐QuIC α‐synuclein positivity in former athletes. Methods: Thirty former athletes with magnetic resonance imaging, neuropsychological testing, and CSF analyzed for phosphorylated tau 181 (p‐tau), total tau (t‐tau), amyloid‐β 42 (Aβ42), and neurofilament light chain (NfL). CSF α‐synuclein was detected using RT‐QuIC. Results: Six (20%) former athletes were α‐synuclein positive. α‐Synuclein positive athletes were similar to α‐synuclein negative athletes on demographics, sports history, clinical features, CSF p‐tau, t‐tau, Aβ42, and NfL; however, had lower grey matter volumes in the right inferior orbitofrontal, right anterior insula and right olfactory cortices. Conclusions: α‐Synuclein RT‐QuIC analysis of CSF may be useful as a prodromal biofluid marker of PD and DLB. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]