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BACKGROUND AND PURPOSE: Advanced MR imaging techniques are critical to understanding the pathophysiology of conditions involving the spinal cord. We provide a novel, quantitative solution to map vertebral and spinal cord levels accounting for anatomic variability within the human spinal cord. For the first time, we report a population distribution of the segmental anatomy of the cervical spinal cord that has direct implications for the interpretation of advanced imaging studies most often conducted across groups of subjects. MATERIALS AND METHODS: Twenty healthy volunteers underwent a T2-weighted, 3T MRI of the cervical spinal cord. Two experts marked the C3–C8 cervical nerve rootlets, C3–C7 vertebral bodies, and pontomedullary junction. A semiautomated algorithm was used to locate the centerline of the spinal cord and measure rostral-caudal distances from a fixed point in the brain stem, the pontomedullary junction, to each of the spinal rootlets and vertebral bodies. Distances to each location were compared across subjects. Six volunteers had 2 additional scans in neck flexion and extension to measure the effects of patient positioning in the scanner. RESULTS: We demonstrated that substantial variation exists in the rostral-caudal position of spinal cord segments among individuals and that prior methods of predicting spinal segments are imprecise. We also show that neck flexion or extension has little effect on the relative location of vertebral-versus-spinal levels. CONCLUSIONS: Accounting for spinal level variation is lacking in existing imaging studies. Future studies should account for this variation for accurate interpretation of the neuroanatomic origin of acquired MR signals.