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Abstract We present a validated protocol for manual segmentation of the thalamus on T1-weighted magnetic resonance imaging (MRI) scans using brain image analysis software. The MRI scans of five normal control subjects were randomly selected from a larger cohort recruited from Lund University Hospital and Landskrona Hospital, Sweden. MRIs were performed using a 3.0T Philips MR scanner, with an eight-channel head coil, and high resolution images were acquired using a T1-weighted turbo field echo (T1 TFE) pulse sequence, with resulting voxel size 1×1×1 mm3 . Manual segmentation of the left and right thalami and volume measurement was performed on 28–30 contiguous coronal slices, using ANALYZE 11.0 software. Reliability of image analysis was performed by measuring intra-class correlations between initial segmentation and random repeated segmentation of the left and right thalami (in total 10 thalami for segmentation); inter-rater reliability was measured using volumes obtained by two other experienced tracers. Intra-class correlations for two independent raters were 0.95 and 0.98; inter-class correlations between the expert rater and two independent raters were 0.92 and 0.98. We anticipate that mapping thalamic morphology in various neuropsychiatric disorders may yield clinically useful disease-specific biomarkers.