Background: The definition of molecular remission by more sensitive methodologies became a cornerstone for stratification and therapeutic decisions in pediatric acute lymphoblastic leukemia (ALL). The role of next-generation sequencing (NGS) for minimal residual disease (MRD) assessment is still under consideration. Methods: Fifty pediatric patients treated with ALL-IC-BFM-2002 protocol were prospectively evaluated for specific clonal rearrangements of immunoglobulin and T cell receptor genes using two PCR-based methods: multiplex PCR following the BIOMED-2 protocol and NGS analysis. The NGS-MRD status and level were determined on day 33 and day 78 from therapy onset with a sensitivity level of 10 -4 and their prognostic value was analyzed after a median follow-up of 4 years. Results: All but one patient with negative NGS-MRD status on day 33 are in clinical remission, emphasizing its predictive value for favorable outcome with event free survival (EFS) exceeding 95%. A total of 29 (58%) patients were NGS-MRD positive on day 33, of which 9 (18%) patients remained positive on day 78. However, only a small percent of the patients with positive NGS-MRD status on day 33 and day 78 relapsed: 21% (6/29) and 33% (3/9), respectively. Conclusion: Positive NGS-MRD status is not a reliable prognostic biomarker in children with ALL and warrants careful consideration in disease stratification.