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CDC25 proteins play a pivotal role in controlling cell proliferation during development and tumorigenesis. The aim of the study is to elucidate the role of CDC25A and CDC25B proteins in retinoblastoma and their association with the clinical and histopathological parameters.One hundred and nine prospective cases of primary enucleated retinoblastomas were included in the present study. Expression of CDC25A and CDC25B proteins was investigated by immunohistochemistry, western blotting and mRNA expression by reverse-transcriptase PCR.Immunohistochemistry showed CDC25A expression in (57/109) 52.29%, whereas CDC25B expressed in (69/109) 63.30% cases. Western blotting confirmed the immunoreactivity results on representative cases. mRNA expression of CDC25A and CDC25B was found in 29/60 (48.33%) and 35/60 (58.33%) cases, respectively. Expression of CDC25A and CDC25B showed significant correlation with poor tumour differentiation and tumour invasion (p<0.05). There was a statistically significant difference in the overall survival of patients with CDC25B expression (p=0.0270).Our results suggest that expression of CDC25B may be used as a potential prognostic marker in the pathogenesis of retinoblastoma. These findings demonstrate an important role of CDC25 phosphatase proteins and inhibition of these proteins may have therapeutic potential in retinoblastoma.