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Purpose: This study is aimed to investigate the presence of Human papillomavirus (HPV) DNA in tumors obtained from sporadic retinoblastoma patients. Methods: One hundred six tumor tissues obtained from sporadic RB patients were analyzed for HPV infection by use of both seminested PCR and real-time quantitative PCR. Results: Of 106 RB patients, 55 were male and 51 were female. The mean age at diagnosis was 26.77 ± 15.36 (mean ± Std. dev) months. Almost all patients presented with leukocoria. Molecular investigation by different methods revealed no HPV positivity in any tumor genome. Conclusion: Our study demonstrates no association between HPV and RB, postulating HPV may not be a major risk factor in the etiology of RB.