Pseudoexfoliation Syndrome (PXS) is a disorder characterized by the progressive accumulation of fibrillary extracellular deposits in several ocular tissues. It is an independent risk factor for glaucomatous optic nerve damage. Retinal Nerve Fiber Layer (RNFL) thickness analysis using optical coherence tomography is a documented investigative tool to detect glaucoma at an early stage. The aim was to evaluate and compare RNFL thickness in PXS patients without glaucoma with their age and sex matched healthy controls and to detect the possibility of early glaucomatous damage in patients with RNFL thinning. A total of 100 patients were included, of which 50 were cases (Group A) and 50 were controls (Group B). RNFL thickness of cases and controls were compared using Zeiss Cirrus HD-OCT 500. There were no significant differences between the two groups with respect to mean RFNL thickness in nasal (p=0.129) and temporal quadrants (p=0.925). Mean inferior RNFL thickness was 112.9 ± 21.72 μm in Group A and 120.6 ± 10.35 μm in Group B (p=0.002). The mean thickness of retinal nerve fiber layer in superior quadrant in Group A was 101.6 ± 23.16 μm whereas in group B it was 113.5 ± 13.47 μm. (p< 0.001). The average RFNL thickness in Group A was 85.1 ± 13.99 μm and in Group B it was 88.9 ± 7.01 μm. (p=0.017). There was statistically significant difference in RNFL thickness between cases and controls in inferior and superior quadrants and in global average thickness.