Rhabdomyosarcoma (RMS) is the most commonly diagnosed malignant soft tissue tumor in children and adolescents. Standard therapeutic approaches for RMS treatment involve surgery, chemotherapy and radiotherapy (RT). In particular, the indications and techniques for pediatric RT evolved considerably over the years, resulting in improved cure rates together with the potential for decreased treatment-related morbidity and mortality. Anyway, despite the initial efficacy, 5-year survival rates of RMS patients remain unfavorable. Individuals with metastatic or recurrent disease have a poor clinical outcome, with a 5-year overall survival of about 30%. RMS relapsing is mainly due to the expansion of cancer stem cells (CSCs), which are able to self-renew and possess a high capability to efficiently repair DNA damage. These features render CSCs resistant to conventional therapies, including RT, and the eradication of all CSCs is a requirement for a more effective antineoplastic treatment and an improvement of patient prognosis. So, a deep understanding of the molecular and cellular pathways that comprise the radio-resistance is an urgent clinical need. In the present project, we aim to extend current knowledge on the mechanisms of radio-resistance in parental and highly radio-resistant RMS cells as well as CSCs by performing gene expression analysis of a large number of coding and non-coding genes. A bioinformatic approach will be used to select drivers of important molecular networks to be addressed for experimental validation. Our final aim is to identify new molecules useful both as biomarkers and targets for setting up new treatment approaches or to improve current therapies, mainly in the context of RT. In particular, our in vitro and in vivo studies are committed to continuing research to refine and modernize the use of RT in current and future protocols, with the goal of further improving the cure rates and quality of life of children stricken with RMS cancer.