Retinoblastoma is the most common malignant intraocular tumor of childhood. Drug resistance and relapses are major problems with chemotherapy, which is regarded as the mainstay of globe preserving treatment in retinoblastoma. P-glycoprotein (P-gp) expression has been reported to be associated with chemoresistance and poor prognosis in various malignancies. We analyzed P-gp expression in retinoblastoma specimens, enucleated either primarily or after neoadjuvant chemotherapy by immunohistochemistry and immunoblotting, and correlated with the histopathological findings. Variables were statistically analyzed by Fischer's exact and chi-square tests. Tumor tissues were collected from enucleated eyes of 24 children. Fifteen of these were primarily enucleated (group I), and nine (group II) had received chemotherapy prior to enucleation. P-gp was expressed in 4/15 (26.7 %) eyes in group I and in 5/9 (55.6 %) eyes in group II. P-gp was highly expressed in group II as compared to group I. There was no correlation between P-gp expression and tumor differentiation, invasion, or laterality. In conclusion, there was markedly high expression of P-gp in eyes with retinoblastoma enucleated after chemotherapy. This may possibly play a role in chemoresistance or it may be that chemotherapy might have induced high expression. These findings may have important implications for the treatment of retinoblastoma patients but need further prospective investigations in a larger patient population.