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Context: Retinal nerve fibre layer (RNFL) being unmyelinated, RNFL thickness (RNFLT) provides direct evidence of axonal loss in optic neuritis (ON) and may prove to be a valuable diagnostic and prognostic tool in the development of multiple sclerosis (MS). Aims: Assessment of RNFLT by OCT3 in patients with recent onset retrobulbar ON, comparison of obtained values based on presence or absence of the periventricular white matter lesion (PVWML) on magnetic resonance imaging (MRI), comparison with the RNFLT of a control group, and analysis of the obtained results. Settings and Design: Cross-sectional study. Materials and Methods: Eighteen patients with <=3 weeks onset clinically diagnosed retrobulbar ON between 19 and 55 years were included. RNFLT was assessed on presentation by OCT3 for both the eyes. MRI of brain and spinal cord with gadolinium enhancement was also performed in all patients, after adequate medical clearance. Patients were divided into: group A (10 patients) with no MRI-proven PVWML and group B (8 patients) with one or more PVWML, operationally deemed to have MS. RNFLT analysis was also done in 18 age-matched controls who were assigned to group C. Statistical Analysis Used: ANOVA and Student′s t test. Results: ON eyes in group B had thinnest RNFLT (average temporal = 39.75 μ), followed by group A (average temporal = 44.3 μ), and finally by group C (average temporal-OU = 80.78 μ). Conclusions: Our study shows that patients with ON, irrespective of detection of PVWML on MRI, have thinner RNFL compared to age-matched controls (P < 0.001). The average RNFLT values are lowest in patients with PVWML. The aspect of RNFL thinning in non-ON eyes should be further studied as a possible subclinical indicator of MS.