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PURPOSE Transvitreal and transscleral needle biopsy can result in complications including vitreous hemorrhage and retinal detachment. This study evaluated a technique using 25-gauge vitrectomy as an adjunct to needle biopsy immediately before brachytherapy to minimize these complications and preserve good visual acuity. DESIGN Retrospective, observational case series. PARTICIPANTS Fifty-seven consecutive eyes of 57 patients with treatment-naïve medium choroidal melanomas without extraocular extension from July 2012 through September 2015. METHODS Fifty-seven consecutive eyes of 57 patients with a clinically diagnosed choroidal melanoma underwent complete 25-gauge posterior vitrectomy followed by transvitrector port fine-needle aspiration biopsy of the tumor immediately before implantation of a radioactive iodine 125 plaque as treatment for the tumor. Cytopathologic analysis was not performed on the tumor aspirates in this study. MAIN OUTCOME MEASURES Best-corrected postoperative visual acuity, postoperative complications of the reported technique, implantation tumor development, local tumor recurrence, presence of metastatic disease after surgery, and sufficiency of the tumor aspirates obtained by the reported technique for successful gene expression profile testing and prognostic classification. RESULTS Mean preoperative and postoperative visual acuities were similar (20/60 vs. 20/80, respectively). Mean tumor thickness was 5.0 mm (range, 2.5-10 mm) and mean tumor basal diameter was 13.1 mm (range, 7-22 mm). Only 1 of 57 eyes (1.8%) showed a transient vitreous hemorrhage, biopsy yield was 100% for genetic analysis, and no patients showed recurrence or implantation tumor at the vitrector site. CONCLUSIONS Combined 25-gauge posterior vitrectomy and 25-gauge trans-vitrector port needle aspiration biopsy immediately before brachytherapy is excellent for obtaining tumor aspirate for gene expression profiling while controlling for hemostasis, resulting in few complications.