BACKGROUND: Currently, transvitreal fine-needle aspiration biopsy is the most widely used tissue biopsy technique in cases of suspected intraocular lymphoma due to its relative simplicity and low trauma. The small sample produced, however, may be inadequate for diagnostic and prognostic analyses due to mechanical artefacts, insufficient material, or sampling errors. Small case series have demonstrated choroidal biopsy via vitrectomy to be safe and effective. With smaller-gauge vitrectomy instruments, visual recovery is rapid, and post-operative inflammation and conjunctival scarring is minimised. Furthermore, smaller-gauge instrumentation does not appear to affect the diagnostic yield of biopsies for intraocular lymphoma in vitro. We report a case of primary choroidal lymphoma successfully diagnosed with 27-gauge pars plana vitrectomy choroidal biopsy. CASE PRESENTATION: A 72-year-old female presented with a 6-month history of painless blurred vision in her right eye. Fundus examination revealed a large pale choroidal mass centred on the posterior pole with overlying exudative retinal detachment. Enhanced depth imaging optical coherence tomography revealed a markedly thickened choroid with an undulating appearance. B-scan ultrasonography demonstrated diffuse, smooth thickening of the choroid, and retrobulbar extrascleral hypoechoic nodules. A 27-gauge pars plana vitrectomy was performed and choroidal biopsy taken. Histopathologic, immunohistochemical, and flow cytometry studies confirmed a diagnosis of extranodal marginal zone B-cell lymphoma. Systemic workup found no evidence of systemic lymphoma. As such, the patient was diagnosed with primary choroidal lymphoma. She underwent intensity-modulated external beam radiotherapy with subsequent resolution of disease. CONCLUSIONS: Primary choroidal lymphoma can be safely and effectively diagnosed via 27-gauge vitrectomy choroidal biopsy.