Purpose: To evaluate the reproducibility of nerve fiber layer (NFL) thickness measurements by optical coherence tomography (OCT) in individuals with silicone oil-filled eyes. Methods: Eighteen patients who had undergone pars plana vitrectomy and silicone oil tamponade for retinal detachment were enrolled in a prospective, case-controlled clinical study. Each patient underwent OCT measurement of NFL thickness. Five repetitions of a series of scans on five separate occasions within a 0.5-month period were performed. Each eye was scanned at two different nerve head programs [radius (R) = 1.5 and R = 1.73]. The contralateral healthy eye was used as control. For each option (R = 1.5 and R = 1.73) and region (superior, inferior, temporal, nasal and overall mean), variance components and intraclass correlation coefficients (ICCs) were determined using repeated-measures regression. In these models, NFL thickness, as measured by OCT, was considered to have three variance components: intersubject, intervisit (within subject, between dates), and intravisit (within subject, within date). The ICC (intersubject variance/total variance) was used as a measure of reliability. Results: The contralateral healthy eye provided a higher degree of reproducibility than the silicone oil-filled eye (p = 0.0001). Reproducibility was higher in a given eye on a given visit than from visit to visit. Reproducibility as measured by ICCs was as follows: R = 1.5, 0.54/0.30 (control eyes/silicone oil-filled eyes); R = 1.73, 0.51/0.30. Conclusions: Reproducibility results for OCT measurement of NFL thickness are different in healthy eyes and silicone oil-filled eyes. NFL thickness measurement is not reliable in silicone oil-filled eyes.