Background Optical coherence tomography (OCT) has become a very useful tool to study in vivo different ocular structures and to improve differential diagnosis and management of many ocular pathologies. This study aims to identify pterygium alterations that trigger characteristic OCT images, and analyze if this pattern correctly demarcates lesion boundary. Methods Thirty-two patients, 22 men, and ten women, aged between 26 and 56 (mean age 40.5±6.9) with symptomatic primary pterygium were recruited. After excision, lesion images were obtained by high-definition OCT. Specimens were stained with hematoxylin–eosin (H&E), antivimentin for all mesenchymal origin cells and altered limbal basal cells, CD45 for lymphocyte and macrophage cells, CD1a for Langerhans cells, and S100 for melanocyte and Langerhans cells. Results The typical OCT wedge-shape hyperreflective mass was evident only by vimentin antibody and included, mainly, fibroblasts but also immune cells (verified by CD45) in a rich network of collagen fibers. The mass apex, often extended centripetally as a thin subepithelial line, hyperreflective by OCT, was formed by a row of fibroblasts under an apparently intact Bowman’s layer, as vimentin samples revealed. Hyperreflective epithelium overlying the mass showed a great number of vimentin-positive infiltrated cells such as melanocytes, Langerhans cells, and lymphocytes (identified by the other biomarkers). H&E staining revealed the presence of goblet cells. Nevertheless, only vimentin staining revealed the presence of altered basal cells above partially dissolved or apparently intact Bowman’s layer, coinciding in this last case with the fibroblast subepithelial line. In most of the cases (72 %), the altered cells occupied a basal segment shorter than the fibroblast subepithelial line but in some specimens, these cells exceeded the fibroblast line length. Conclusions This study demonstrated the great visual accordance between pterygium OCT images and vimentin staining. Alteration in collagen arrangement, infiltration of inflammatory cells, and fibroblast subepithelial line in the lesion apex were the main histological changes responsible for the anomalous hyperreflectivity of the OCT pattern. By contrast, altered basal cells located in the basal epithelial layer of the pterygium head could not be detected by OCT, which might generate lesion size underestimation.