To correlate spectral-domain optical coherence tomography (SDOCT) criteria and clinical data with pathology of the vitreomacular interface (VMI) in eyes with diabetic macular edema (DME). Retrospective cross-sectional study and laboratory investigation. We included specimens of 27 eyes of 26 patients with center-involved DME that underwent vitrectomy with peeling of the internal limiting membrane (ILM). Selection of specimens was consecutive and in retrospect using our register of the Vitreoretinal Pathology Unit. Clinical data and SDOCT examinations were correlated to immunocytochemistry and transmission electron microscopy. Classification of DME comprised sponge-like diffuse retinal thickening, cystoid macular edema, and serous retinal detachment. VMI was evaluated for presence of epiretinal membrane (ERM) and thickened vitreous cortex (tVC). ERMs and tVC were found in all DME types. Diffuse DME showed tVC more often than cystoid DME. Hyalocytes, contractile myofibroblasts, glial cells, matrix metalloproteinases-2 and -9, and collagen type I, II, and III were positive tested irrespective of DME type. There were no significant cell fragments at the retinal side of the ILM. Visual acuity improved in the majority of cases and macular thickness decreased significantly during mean follow-up of 17 ± 10months. All eyes presented pathologic VMI changes irrespective of the OCT classification of DME type or presence of ERM. Composition of fibrocellular membranes at the VMI indicated remodeling of vitreous cortex and transdifferentiation of hyalocytes into myofibroblasts. Our findings might argue for an early surgical intervention in eyes with DME irrespective of the presence of traction formation imaged by SDOCT.