BACKGROUND: The recent International Parkinson and Movement Disorder Society diagnostic criteria for multiple system atrophy (MDS-MSA) have been developed to improve diagnostic accuracy although their diagnostic properties have not been evaluated. OBJECTIVES: The aims were to validate the MDS-MSA diagnostic criteria against neuropathological diagnosis and compare their diagnostic performance to previous criteria and diagnosis in clinical practice. METHODS: Consecutive patients with sporadic, progressive, adult-onset parkinsonism, or cerebellar ataxia from the Queen Square Brain Bank between 2009 and 2019 were selected and divided based on neuropathological diagnosis into MSA and non-MSA. Medical records were systematically reviewed, and clinical diagnosis was documented by retrospectively applying the MDS-MSA criteria, second consensus criteria, and diagnosis according to treating clinicians at early (within 3 years of symptom onset) and final stages. Diagnostic parameters (sensitivity, specificity, positive/negative predictive value, and accuracy) were calculated using neuropathological diagnosis as gold standard and compared between different criteria. RESULTS: Three hundred eighteen patients (103 MSA and 215 non-MSA) were included, comprising 248 patients with parkinsonism and 70 with cerebellar ataxia. Clinically probable MDS-MSA showed excellent sensitivity (95.1%), specificity (94.0%), and accuracy (94.3%), although their sensitivity at early stages was modest (62.1%). Clinically probable MDS-MSA outperformed diagnosis by clinicians and by second consensus criteria. Clinically established MDS-MSA showed perfect specificity (100%) even at early stages although to the detriment of low sensitivity. MDS-MSA diagnostic accuracy did not differ according to clinical presentation (ataxia vs. parkinsonism). CONCLUSIONS: MDS-MSA criteria demonstrated excellent diagnostic performance against neuropathological diagnosis and are useful diagnostic tools for clinical practice and research. © 2023 International Parkinson and Movement Disorder Society.