BACKGROUND/AIMS Diagnostic pars plana vitrectomy is a useful technique in the diagnosis of intraocular lymphoma (IOL); however, the role of transconjunctival sutureless vitrectomy (TSV) has not been fully explored for this indication. The purpose of this study was to review our experience with 25-gauge TSV for the diagnosis of IOL. METHODS Patients who underwent 25-gauge TSV for the diagnosis of IOL (primary, secondary or recurrent) from two tertiary referral centres were reviewed. Demographic data and underlying medical conditions were reviewed. Preoperative and postoperative visual acuities (VA) and ophthalmic examination data were assessed. Cytopathology, flow cytometry, cytokine and gene rearrangement studies were assessed. RESULTS Twelve patients underwent 25-gauge diagnostic TSV with a median follow-up time of 37 weeks. B-cell or T-cell IOL was diagnosed based on cytology in 3/12 patients (25%, 95% CI 8.9 to 53.2%) and in eight patients (67%, 95% CI 39.1 to 86.1%) using adjunctive diagnostic testing. VA stabilised or improved in 11 eyes (92%). Mean VA improved from 20/95 to 20/66 (p=0.055, paired t test). CONCLUSIONS 25-Gauge TSV is safe and effective for obtaining vitreous specimens for the evaluation of IOL. The availability of expert ophthalmic pathological consultation, flow cytometry, cytokine evaluation and gene rearrangement studies were essential to the diagnosis.