Corpus callosum atrophy is a favorable imaging biomarker in MS. Manual measurements of the corpus callosum are considered the best current standard, but their repeatability and reproducibility are uncertain. FreeSurfer is an automatic software that can volumetrically measure the corpus callosum. Using a representative cohort of 9 MS patients, scanned twice with repositioning on 3 different MRI scanners, we compared the manual and automatic measurements of corpus callosum. We found the longitudinal FreeSurfer method to be the most precise method. Thus, we recommend that this method be used in future studies of measuring corpus callosum atrophy in patients with MS.