Spectral domain optical coherence tomography-based analysis of retinal architecture after internal limiting membrane peeling for macular hole surgery. Prospective, interventional study. Fifty eyes underwent the surgical procedure with minimum internal limiting membrane peel of 3 mm diameter. Automatic segmentation software was used to assess individual layers preoperatively and postoperatively, 1.5 millimeters medial and lateral to fovea at 3 months postoperative visit. Main outcome measures were final central macular thickness and variation in individual retinal layer thickness. Mean central macular thickness postoperatively was 201 microns. Retinal thickening was observed, 1.5 mm medial to fovea (P < 0.01) with significant increment in thickness of inner nuclear layer (P < 0.01), whereas significant retinal thinning was observed 1.5 mm temporal to fovea (P < 0.01) with maximum thinning observed in ganglion cell layer (P < 0.01). Ganglion cell and inner plexiform were the only layers to demonstrate thinning on both sides of the fovea. External limiting membrane (ELM), inner segment outer segment junction (ISOS) and cone outer segment tips zone (COST) restoration was seen in 100%, 69%, and 17% of eyes. Best corrected visual acuity (BCVA) improved significantly with mean postoperative BCVA of 0.7logMAR (6/30 Snellens equivalent) (P < 0.001). Anatomical closure rate of 72% was achieved (P < 0.001). Internal limiting membrane peel is associated with significant alteration in inner retinal architecture, especially in ganglion cell layer, which can adversely influence functional outcome of the surgery and makes it imperative to avoid peeling internal limiting membrane over a larger surface area.