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To develop a method based on identification of the widest region of the surgical limbus that can yield quick and accurate orientation of excised human donor corneas.Corneoscleral tissue from donors 49 to 75 years old was marked at the temporal sclera at the time of recovery. Digital images obtained from 20 corneas stored in viewing chambers, retroilluminated and viewed from the endothelial side, were used to quantify the per-degree radial width of the surgical limbus, defined as the distance from the scleral spur to clear cornea. To evaluate differences in radial width among regions, measurements were compared with the intracorneal mean limbal width, and a per-degree z-score was calculated by averaging among corneas. Using images of corneas with the temporal mark masked and the sidedness known, 6 observers were subjected to a blinded trial of 10 corneas to determine the central point of the widest limbal region of each cornea.Compared with the intracorneal mean, the mean radial width of the surgical limbus was greatest in the superior quadrant, and the difference compared with the inferior, nasal, and temporal quadrants was significant (P < 0.0001). The superior region was identified with 100% accuracy in blinded trials. The average absolute difference between the predicted and actual central point of the superior limbus was 9.75 ± 0.30 degrees.The radial width of the surgical limbus is greatest in the superior region of the cornea and can be used as a diagnostic feature to orient donor corneal tissue.