OBJECTIVE: To measure changes in the thickness of the retinal nerve fiber layer in normal and early glaucomatous dogs with scanning laser polarimetry. ANIMALS STUDIED: A total of 45 eyes, 32 normal and 13 glaucomatous eyes, of American Cocker Spaniels with primary glaucoma were used. All eyes were evaluated through a complete neuro-ophthalmic examination, tonometry, gonioscopy, slit-lamp biomicroscopy, and indirect ophthalmoscopy prior to enucleation. METHODS: The retinal nerve fiber layer thickness was measured in anesthetized animals with scanning laser polarimetry (Nerve fiber analyzer, GDx; Laser Diagnostic Technologies, LTD, San Diego, CA, USA). Glaucomatous eyes retained some vision at the time of this study. RESULTS: The mean +/- SD of the retinal nerve fiber layer thickness was 141.69 +/- 18 microm for normal dogs and 105.08 +/- 23.86 microm for visual glaucomatous dogs. The average retinal nerve fiber layer thickness in the superior and inferior retinal quadrants was 148.03 +/- 8.5 and 141.06 +/- 8.73 microm, respectively, for normal dogs, and 106.61 +/- 25.77 and 107.08 +/- 24.99 microm in the superior and inferior retinal quadrants, respectively, for glaucomatous dogs. The superior to nasal retinal nerve fiber layer thickness ratio was 1.45 for normal dogs and 1.26 for visual glaucomatous dogs. CONCLUSIONS: Using scanning laser polarimetry it was possible to detect changes in retinal nerve fiber layer thickness in glaucomatous dogs at early stages of the disease. Therefore, this instrument has the potential to improve the clinical management of canine glaucoma by detecting progressive changes to the retinal nerve fiber layer.