Recent neuropathologically confirmed clinical data suggest that the midbrain-to-pons ratio, as calculated from conventional brain MRI, has high specificity and sensitivity for the diagnosis of progressive supranuclear palsy (PSP). Here, we aimed to replicate these findings in an independent autopsy-confirmed cohort of 6 PSP patients and 23 non-PSP patients. Patients with confirmed PSP had clearly lower midbrain-to-pons ratios compared to non-PSP patients (p < 0.0001). All non-PSP patients had midbrain-to-pons ratios higher than 0.50, whereas all but one PSP patient had a ratio lower than 0.50. The positive predictive value (PPV) of the ratio (< 0.50) was 100% and the negative predictive value (NPV) was 95.8 %. The results of this second autopsy-confirmed sample confirm that midbrain-to-brain ratios constitute reliable and clinically useful estimates of diagnostic midbrain atrophy in relation to PSP pathology.