Purpose To evaluate the utility of endothelial/Descemet membrane complex (En/DM) characteristics in diagnosing corneal graft rejection. Design Diagnostic reliability study. Methods One hundred thirty-nine eyes (96 corneal grafts post penetrating keratoplasty or Descemet stripping automated endothelial keratoplasty: 40 clear, 23 actively rejecting, 24 rejected, and 9 nonimmunologic failed grafts; along with 43 age-matched control eyes) were imaged using high-definition optical coherence tomography. Images were used to describe En/DM and measure central corneal thickness (CCT) and central En/DM thickness (DMT). En/DM rejection index (DRI) was computed to detect the relative En/DM thickening to the entire cornea. Results In actively rejecting grafts, DMT and DRI were significantly greater than controls and clear grafts (28, 17, and 17 μm and 1.5, 1 and 1, respectively; P < .001). Rejected grafts had the highest DMT and DRI compared to all groups (59 μm and 2.1; P < .001). DMT and DRI showed excellent accuracy, significantly better than that of CCT, in differentiating actively rejecting from clear grafts (100% and 96% sensitivity; 92.5% and 92.5% specificity), actively rejecting from rejected grafts (88% and 83% sensitivity; 91% and 83% specificity), and nonimmunologic failed from rejected grafts (100% and 100% sensitivity; 88% and 100% specificity). DMT correlated significantly with rejection severity (P < .001). Conclusions In corneal grafts, in vivo relative thickening of the En/DM is diagnostic of graft rejection as measured by DMT and DRI. These indices have excellent accuracy, sensitivity, and specificity in detecting graft immunologic status, superior to CCT. DMT is a quantitative index that correlates accurately with the severity of rejection.