Objective: To evaluate the retinal nerve fiber layer (RNFL) thickness in patients with unilateral pseudoexfoliation syndrome (PXS) without glaucoma by using optical coherence tomography (OCT). Methods: 22 patients with unilateral PXS were evaluated. Group 1 included the eyes with the pseudoexfoliation, group 2 included the fellow eyes; 18 age-matched normal control eyes were assessed in group 3. The mean and segmental RNFL thickness in eyes with pseudoexfoliation and their fellow eyes in patients with unilateral PXS was compared to age-matched control subject eyes. Results: The RNFL in patients with PXS were significantly thinner than controls in all quadrants except the nasal quadrant with regard to segmental analysis (p < 0.05). This RNFL loss was apparent at 7, 10 and 11 o’clock of the PXS eyes with regard to clock hour position (p < 0.05). In the fellow eyes, no significant difference in RNFL measurement was found except the temporal quadrant when compared with the controls. In the analysis with regard to clock hour position, no significant reduction in RNFL thickness was found except in the 11-o’clock segment. In PXS eyes, RNFL thicknesses at the inferior quadrant and the 1-, 2- and 5-o’clock segments were significantly lower than in non-PXS eyes (p < 0.05). Conclusions: This study suggests that PXS without glaucoma may be associated with a thinner RNFL compared with those of age-matched control subjects and non-PXS fellow eyes. Further studies are needed to clarify the relationship between the decrease in RNFL thickness and the development of glaucomatous damage in eyes with pseudoexfoliation.