Although early detection is critical, diagnosing vitreoretinal lymphoma (VRL) remains difficult. We sought to assess the potential diagnostic value of spectral-domain optical coherence tomography (SD-OCT) in VRL.We reviewed the clinical records and pre-treatment SD-OCT images of biopsy-confirmed VRL and uveitis patients, with primary involvement of the sub-retinal pigment epithelium (RPE) and the outer retina, including acute syphilitic posterior placoid chorioretinitis (ASPPC), chronic stage sympathetic ophthalmitis (SO), and idiopathic multifocal choroiditis (MFC).We included 45 eyes of 45 VRL patients and 40 eyes of 40 uveitis patients (17 ASPPC eyes, eight chronic SO eyes, and 15 MFC eyes). On SD-OCT, lymphoma cell infiltration was observed in various retinal layers, most commonly in the sub-RPE (80%) and sub-retinal space (62%). Highly sensitive features for VRL as compared to uveitis included vitreous cells (93%), focal hyper-reflective sub-retinal infiltration (51%), and diffuse RPE elevations (56%). The features strongly specific for VRL included preretinal deposits (92.5%), intra-retinal infiltration (except the incomplete vertical hyper-reflective type, 100%), banded hyper-reflective sub-retinal infiltration (90%), and confluent RPE detachments (100%). We identified an approach to VRL diagnosis based on these SD-OCT findings: 1) two highly sensitive features plus one strongly specific feature; or 2) one highly sensitive feature plus two strongly specific features, demonstrated a sensitivity of 80% and specificity of 95% for VRL.SD-OCT may enable the detection of detailed lymphoma infiltration characteristics and provide significant supplemental value for VRL diagnosis, particularly when combining highly sensitive and specific VRL-associated SD-OCT features. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.