Background: Neuroblastoma (NB) is the most common extracranial pediatric solid tumor with a highly variable clinical course, ranging from spontaneous regression to life-threatening disease. Survival rates for high-risk NB patients remain disappointingly low despite multimodal treatment. Thus, there is an urgent clinical need for additional biomarkers to improve risk stratification, treatment management, and survival rates in children with aggressive NB. Results: Using gene promoter methylation analysis in 48 neuroblastoma tumors with microarray technology, we found a strong association between survival and gene promoter hypermethylation (P = 0.036). Hypermethylation of 70 genes significantly differentiated high-risk survivor patients from those who died during follow-up time. Sixteen genes with relevant roles in cancer biology were further validated in an additional cohort of 83 neuroblastoma tumors by bisulfite pyrosequencing. High promoter methylation rates of these genes were found in patients with metastatic tumors (either stage metastatic (M) or metastatic special (MS)), 18 months or older at first diagnosis, MYCN amplification, relapsed, and dead. Notably, the degree of methylation of retinoblastoma 1 (RB1) and teratocarcinoma-derived growth factor 1 (TDGF1) predicts event-free and overall survival independently of the established risk factors. In addition, low RB1 mRNA expression levels associate with poor prognosis suggesting that promoter methylation could contribute to the transcriptional silencing of this gene in NB. Conclusions: We found a new epigenetic signature predictive for NB patients' outcome: the methylation status of RB1 and TDGF1 associate with poorer survival. This information is useful to assess prognosis and improve treatment selection.