Purpose To determine whether limbal epithelial stem cells (LESCs) repopulate the site harvested for limbal autograft transplantation (LAT), the expression of LESCs markers was evaluated in bioptic specimens obtained from the donor area 12 months or more after surgery. Design Interventional case series. Participants Patients who underwent LAT for unilateral acquired limbal stem cell deficiency after chemical burn. Methods Corneal limbal explants were obtained from 2 sites, the harvested area and the untouched control area, in the donor eyes of 6 patients who previously underwent LAT for unilateral acquired limbal stem cell deficiency after chemical burn. Limbal epithelial stem cells were isolated, and cellular, immunohistochemistry, and histologic parameters were assessed to compare differences between LESCs isolated from harvested or control sites. Main Outcome Measures Presence of LESCs 1 year or more after LAT. Results Specific markers (p63, Ki67, K12), percentage of LESCs, cell doubling, and number of passages in culture did not differ significantly between harvested and control sites. However, the distinctive structure of the palisades of Vogt was found only in 2 of 6 harvested sites. Conclusions Limbal epithelial stem cells repopulate the donor site as early as 1 year after limbus removal for LAT. Autologous transplantation of conjunctiva and limbus are safe procedures and can be performed in cases that cannot be treated by simple grafting of LESCs cultured ex vivo.