Objective: The aim of the present study was to assess the size of the third ventricle in a sample of patients with multiple sclerosis (MS) affected by chronic cerebrospinal venous insufficiency (CCSVI), versus size of the third ventricle in a healthy control group. Background: CCSVI, a new nosological vascular pattern, has recently been associated with MS. Methods: We enrolled 33 patients affected by MS (three in the primary progressive clinical course, 23 in the relapsing–remitting clinical course, and seven in the secondary–progressive clinical course). All patients had been affected by CCSVI and were tested using an echo color Doppler (ECD) imaging unit. The group of 33 affected by both MS and CCSVI (MS–CCSVI) was composed of 19 females and 14 males with a mean age of 40±10 years, from a minimum age of 20 years to a maximum age of 66 years, with a median of 40 years and a mode of 32 years. We compared the 33 MS–CCSVI patients with 33 healthy control subjects of similar sex and age. In the MS–CCSVI group, the MS clinical severity was expressed by expanded disability status scale (EDSS) score: light (19 patients, scoring 1, 2, or 3); medium (ten patients with a score of 4, 5, or 6), and severe (four patients with a score of 7, 8, or 9). The average duration of the MS was 10±7 years (from a minimum of 1 to a maximum of 26 years, with a median of 10 years and a mode of 1 year). Results: In the MS–CCSVI group, the third ventricle diameter was 6.2±1.7 mm (from a minimum of 2.5 mm to a maximum of 9.2 mm, with a median of 6.3 mm, and a mode of 6.0 mm). Our data showed that 29 patients (88%) had an increase in third ventricle diameter, whereas only four patients (12%) had physiological size (less than 4 mm) comparable to all healthy control group subjects (27.28%). These results show that the increase in the third ventricle diameter could represent a criterion of positivity of neurological disease in patients with CCSVI.