Glaucoma is a leading cause of irreversible vision loss and is associated with fibrotic changes in two ocular tissues-the optic nerve head (ONH) and trabecular meshwork (TM). We investigated the differences in extracellular matrix components (ECM) including collagen, elastin, transforming growth factor beta-2, type-II receptor (TGFβRII) and Galectin3 (Gal3) in the glaucomatous human eyes to quantify fibrotic changes in ONH and TM. Glaucomatous and control human donor eyes were prepared for chemical and immunological staining to quantify ECM protein expression in the TM and ONH. Chemical staining included: Trichrome (collagen), Vernhoeff-Van Giesen (elastin) and Sirius Red (collagen). Immunohistochemistry was used to determine levels of Gal3 and TGFβ2RII. Quantitative analyses were performed using Image J software. Student's t-test was used to compare groups and Pearson's test was used to determine correlations P-values of 0.05 (or less) were considered statistically significant. Deposition of ECM proteins was elevated in glaucomatous tissues. There was increased collagen (P = 0.0469), Gal3 (P < 0.0001) and TGFβ2RII (P = 0.0005) in the TM of glaucomatous eyes. Likewise, collagen (P = 0.0517) and Galectin3 (P = 0.041) were increased in the ONH glaucomatous eyes. There was a correlation of TGFβRII with Gal3 in the TM (P < 0.0001) and optic nerve (P = 0.0003). The TM and ONH of glaucomatous eyes showed increased expression of ECM proteins supporting a fibrotic pathology. Galectin3 and TGFβ-2R II showed a positive correlation in TM and optic nerve supporting co-localization and suggesting their potential role in the glaucoma fibrotic process. Clin. Anat. 31:1031-1049, 2018. © 2018 The Authors. Clinical Anatomy published by Wiley Periodicals, Inc. on behalf of American Association of Clinical Anatomists.