Background: Retinoblastoma, the most common intraocular pediatric cancer, presents complexities in its genetic landscape that necessitate a deeper understanding for improved therapeutic interventions. This study leverages computational tools to dissect the differential gene expression profiles in retinoblastoma. Methods: Employing an in silico approach, we analyzed gene expression data from public repositories by applying rigorous statistical models, including limma and de seq 2, for identifying differentially expressed genes DEGs. Our findings were validated through cross-referencing with independent datasets and existing literature. We further employed functional annotation and pathway analysis to elucidate the biological significance of these DEGs. Results: Our computational analysis confirmed the dysregulation of key retinoblastoma-associated genes. In comparison to normal retinal tissue, RB1 exhibited a 2.5-fold increase in expression (adjusted p < 0.01), while E2F3 showed a 3-fold upregulation (adjusted p < 0.05). Additionally, novel genes implicated in chemo-resistance, such as ABCB1, were identified with a significant 3.5-fold decrease in expression (adjusted p < 0.001). Furthermore, differential expression of immune response genes was observed, with a subset demonstrating over a 2-fold change (adjusted p < 0.05). These results, validated against independent datasets, yielded a high concordance rate, thereby substantiating the methodological soundness of our study. Conclusions: Our analysis reinforces the critical genetic alterations known in retinoblastoma and unveils new avenues for research into the disease's molecular basis. The discovery of chemoresistance markers and immune-related genes opens potential pathways for personalized treatment strategies. The study's outcomes emphasize the power of in silico analyses in unraveling complex cancer genomics.