The aim of this study was to identify the internal limiting membrane (ILM) and epiretinal membrane (ERM) harvested during macular hole surgery under light microscopy and electron microscopy and to evaluate the difference in specimen loss rate with and without the aid of indocyanine green (ICG) staining. Thirty-two consecutive patients (34 eyes) with stage 2–4 macular holes were included. ERM peeling (macular hole with ERM) or ILM peeling (macular hole without ERM), with or without the use of ICG, was used during macular hole surgery with vitrectomy. There were 26 specimens for pathology; 12 specimens (46%) were lost during processing. ICG staining did not reduce the specimen loss rate. The specimens harvested from intentional ILM peeling displayed a smooth, translucent ILM. The specimens harvested from ERM peeling showed moderate cellular proliferation abutting on the ILM, and all specimens had an ILM. The pathological findings confirmed the clinical observations in all the cases studied. The ILM appeared in all specimens harvested by ERM peeling during macular hole surgery. ICG staining does not alter the specimen loss rate during pathological processing.