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We aimed to study the histopathological and immunohistochemistry features in clinically diagnosed cases of nanophthalmos using light microscopy. This was an observational comparative study. We enrolled four eyes of four consecutive patients with nanophthalmos and visually significant cataract, who underwent cataract surgery with prophylactic posterior sclerostomy. Histological analysis of the excised scleral tissue was done and compared with age-matched cadaver controls between January 2021 and October 2021. Hematoxylin and Eosin (H&E) stains were used for histological analysis, and was further supplemented with immunohistochemistry (IHC) and immunofluorescence (IF) analyses using a simple light microscope. The immunostained sections were analyzed using confocal microscope for the fibronectin expression level. The main outcome measure was demonstration of histological changes of sclera in nanophthalmic eyes undergoing cataract surgery. Light microscopic features of nanophthalmos revealed thick fibers with fraying and lightly stained cores, irregular serrated edges, and randomly interspersed fibroblasts compared to regular arrangement of collagen fibers seen in cadaver controls. Immunohistochemistry analysis with anti-fibronectin antibody showed strong positivity in clustered fibers in nanophthalmos, and less intense diffuse staining in cadaver tissue. Histoclinical correlation was observed in one nanophthalmic scleral tissue with axial length less than 17 mm showing severe disorganization with diffuse collagenization, loss of fibrillary architecture compared to another specimen with axial length more than 17 mm. Simple, cost-effective light microscopy using basic stains was effective in identifying the characteristic histopathological features in nanophthalmic eyes, and this was further highlighted by immunohistochemistry and immunofluorescence analyses.