Background: Lewy Body pathology can be detected by alpha-synuclein-Seed Amplification Assay (αSyn-SAA) in cerebrospinal fluid (CSF) with high sensitivity and specificity in cohort studies. Yet, little is known about the results that can be expected from αSyn-SAA in CSF in samples outside cohort studies as obtained in clinical routine. Objective: This study analyzed the concordance of αSyn-SAA findings in CSF with clinical diagnosis in patients from clinical routine with diverse neurologic and psychiatric conditions. Methods: In this cross-sectional study, CSF from patients who underwent lumbar puncture for therapeutic or diagnostic purposes were tested in αSyn-SAA. Analysis included binary αSyn-SAA findings, data collected during neurological examination, and structured medical history. Results: All 356 participants (Mean Age 67.1 years, SD = 16.2; 55.9% male) were included in the primary analysis, including 90 patients with Parkinsonian syndromes, 139 with predominant cognitive disorders, 25 with other movement disorders, 35 with inflammatory or (para)neoplastic syndromes, and 67 with further diseases. αSyn-SAA was positive in all samples from patients with Parkinson's disease (41), dementia with Lewy bodies (30), pure autonomic failure (4), and in a subset of patients with Alzheimer's disease (13/46), normal pressure hydrocephalus (7/14) and others.ConclusionsαSyn-SAA findings show high concordance with a clinical diagnosis of PD and DLB. Findings are comparable to results from well-characterized cohort studies, supporting potential diagnostic value in future clinical routine. Challenges may result from the fact that αSyn-SAA detect LB co-pathology that is known from neuropathological studies for several neurodegenerative diseases.