Objective To analyze patient management and prognosis after vitrectomy in eyes with unsuspected retinoblastoma. Design Retrospective, noncomparative case series. Participants Eleven consecutive patients who had undergone vitrectomy on an eye with unsuspected retinoblastoma. Main outcome measures The two main outcome measures were ultimate patient management and the development of retinoblastoma metastasis. Results Of more than 900 consecutive patients with retinoblastoma managed on the Ocular Oncology Service at Wills Eye Hospital in Philadelphia, 11 (1%) had prior vitrectomy in an eye with viable tumor before referral to us for suspected retinoblastoma. The main preoperative diagnoses included vitreous hemorrhage in seven patients (64%), toxocariasis in two patients (18%), toxoplasmosis in one patient (9%), and endophthalmitis in one patient (9%). In no case was retinoblastoma suspected before vitrectomy. The mean patient age at vitrectomy was 6 years. Retinoblastoma was later suspected during vitrectomy in two patients (18%), on cytologic examination of the vitrectomy specimen in eight patients (73%), and after referral in one patient (9%). The mean interval between vitrectomy and referral to us was 23 days. On examination, the globe was classified as Reese-Ellsworth group Vb in all 11 patients (100%). Anterior chamber tumor cells were clinically visible in four eyes (36%), hyphema in two eyes (18%), and iris neovascularization in two eyes (18%). Retinoblastoma cells were visualized in the vitreous in seven eyes (64%) and not visualized in four eyes (36%) that had vitreous blood. Enucleation was necessary in all 11 patients (100%). Adjuvant treatment was delivered in 10 patients (91%), using orbital radiotherapy in nine patients (82%) and chemotherapy in nine patients (82%). Histopathologic evidence of retinoblastoma invasion was documented in the episclera (two eyes; 18%), anterior chamber (seven eyes; 64%), iris (five eyes; 45%), ciliary body (five eyes; 45%), choroid (three eyes; 27%), and optic nerve (four eyes; 36%; prelaminar, two eyes; postlaminar, two eyes). The vitrectomy ports, Tenon’s fascia, cut end of the optic nerve, and orbit were free of tumor. Of the 10 patients who received prophylactic chemotherapy, radiotherapy, or both in addition to enucleation for prevention of retinoblastoma metastasis, none (0%) experienced metastasis or orbital recurrence during the mean follow-up of 7 years (range, 0.2–24 years) from the time of retinoblastoma diagnosis. However, one patient was referred to us after the development of metastatic retinoblastoma, and despite aggressive chemotherapy and radiotherapy after enucleation, died 24 months later. Conclusions Retinoblastoma may present with atypical features such as vitreous hemorrhage or signs of vitreous inflammation, particularly in older children. Vitrectomy should be avoided in these cases until the possibility of underlying retinoblastoma is excluded. If vitrectomy is performed in an eye with unsuspected retinoblastoma, enucleation combined with adjuvant chemotherapy, radiotherapy, or both without delay is advised to prevent systemic tumor dissemination.