This viewpoint evaluates neuropathological evidence to assess the performance and limitations of current biomarker-based frameworks for α-synucleinopathies. Data from autopsy-confirmed and clinical studies indicate that cerebrospinal fluid and skin α-synuclein seeding amplification assays (SAA) achieve high pooled sensitivity and specificity for distinguishing α-synucleinopathies from controls. However, SAA sensitivity decreases when pathology is restricted to the brainstem or amygdala, and current assays cannot reliably identify the affected brain region, cell type, or discriminate co-existing proteinopathies. Peripheral tissue α-synuclein detection shows inconsistent correlation with brain pathology, reflecting variability in sampling, processing, and cohort characteristics. Evidence demonstrates that α-synuclein strain differences influence seeding properties and may permit discrimination between Parkinson’s disease and multiple system atrophy, but protocols require standardisation and validation. Neuropathological data highlight the need for combined biomarker strategies, including molecular imaging and fluid-based proteomics, to improve subtype classification and prognostic accuracy. Priority recommendations include harmonised assay development, expanded autopsy-based correlation, and inclusion of diverse community-based cohorts to ensure biological definitions reflect the full spectrum of disease.