Cervical sagittal alignment is a critical component of successful surgical outcomes. Unrecognized differences in intervertebral alignment between supine and upright positions may affect clinical outcomes;​ however, these differences have not been quantified. Sixty-four patients scheduled to undergo one or two-level cervical arthrodesis for symptomatic pathology from C4–C5 to C6–C7, and forty-seven controls were recruited. Upright sagittal alignment was obtained through biplane radiographic imaging and measured using a validated process with accuracy better than 1° in rotation. Supine alignment was obtained from computed tomography scans. Coordinate systems used to measure supine and upright alignment were identical. Distances between adjacent bony endplates were measured to calculate disc height in each position. For both patients and controls, the C1–C2, C2–C3, and C3–C4 motion segments were in more lordosis when upright as compared with supine (all p < 0.001). However, the C4–C5, C5–C6, and C6–C7 motion segments were in less lordosis when upright as compared with supine (all p ≤ 0.004). There was an interaction between group and position at the C1–C2 (p = 0.002) and C2–C3 (p = 0.001) motion segments, with the controls demonstrating a greater increase in lordosis at both motion segments when moving from supine to upright. The results indicate that cervical motion segment alignment changes between supine and upright positioning, those changes differ among motion segments, and cervical pathology affects the magnitude of these changes. Clinical Significance:​ Surgeons should be mindful of the differences in alignment between supine and upright imaging and the implications they may have on clinical outcomes.