Background Retinoblastoma (Rb) is the most prevalent intraocular pediatric malignancy of the retina. Significant genetic factors are known to have a role in the development of Rb. Methods Here, we report the mutation status of 4813 clinically significant genes in six patients with noncarrier of RB1 gene mutation and having normal RB1 promoter methylation from three families having higher risk for developing Rb in the study. Results A total of 27 variants were detected in the study. Heterozygous missense variants c.1162G > A (p.Gly388Arg) in the FGFR4 gene; c.559C > T (p.Pro187Ser) in the NQO1 gene were identified. The family based evaluation of the variants showed that the variant, c.714T > G (p.Tyr238Ter), in the CLEC7A gene in first family; the variant, c.55C > T (p.Arg19Ter), in the APOC3 gene and the variant, c.1171C > T (p.Gln391Ter), in the MUTYH gene in second family; and the variant, c.211G > A (p.Gly71Arg), in the UGT1A1 gene in the third family, were found statistically significant (p < 0.05). Conclusion This study might be an important report on emphazing the mutational status of other genes in patients without RB1 gene mutations and having high risk for developing Rb. The study also indicates the interaction between the retinoic acid pathway and Rb oncogenesis for the first time.