Intrachromosomal Amplification of chromosome 21 (iAMP21) is an independent outcome indicator in B-cell Precursor ALL (BCP-ALL). IAMP21 is characterized by multiple RUNX1 copies in blast cells, and comprises 2% of children with BCP-ALL. It was first detected during routine screening for ETV6-RUNX1 fusion by FISH. This disease features protocol-dependent outcomes. For example, in pediatric patients, iAMP21-ALL presents poor outcomes when standard therapy is used. Although, when children receive intensive treatment, they usually show lower relapse risk and improved survival. Thus, the early detection of iAMP21 is crucial to guide therapeutic strategies. In this regard, FISH probes may provide a rapid and effective screening, being a valuable ally for banding cytogenetics. In this work, we describe the clinical and molecular data of four pediatric patients with BCP-ALL iAMP21, showing the importance of RUNX1-RUNX1T1 and ETV6-RUNX1 FISH probes in screening this high-risk entity. This collaborative study included four children with BCP-ALL iAMP21. The peripheral blood and Bone Marrow (BM) samples were referred to the cytogenetics laboratory at the Brazilian National Cancer Institute between 2013 and 2021. We performed G-banding and FISH experiments in interphase nuclei and metaphase spreads. We used commercial Locus-Specific (LSI) ETV6-RUNX1, RUNX1-RUNX1T1, and centromeric 13/21 FISH probes. Using the FISH technique with LSI ETV6-RUNX1 and RUNX1-RUNX1T1 probes, we identified the derivative chromosome 21 and the precise number of RUNX1 signals for each patient. It also helped disclose whether the RUNX1 amplifications were intra- and or extrachromosomal. The intrachromosomal amplification of chromosome 21 is an uncommon high-risk chromosomal aberration in pediatric B-ALL. iAMP21 can be elusive, sometimes escaping detection by banding cytogenetics. Thus, confirmation by molecular techniques is necessary. Besides, recent studies suggest a relapse risk associated with therapy intensity, emphasizing the importance of a rapid and early detection of iAMP21 for appropriate risk stratification and therapeutic intervention. The FISH approach used through our experimental design has proved efficient in the early detection of iAMP21 in all patients. Importantly, it helped determine the diagnosis of iAMP21 48 hours after initial diagnosis. Our approach can also be valuable in monitoring patients for Minimal Residual Disease (MRD). According to the literature, those who continuously present MRD should be eligible for BM transplantation. In summary, the flexibility of FISH probes enabled the rapid detection of this high-risk neoplasm. Besides, the precise characterization of iAMP21, including the aberrant chromosomes 21 and extra RUNX1 signals, was important to guide treatment strategies and the monitoring of patients. In addition, we reinforce the importance of investigating the heterogeneity of iAMP21 marker chromosomes, their molecular complexity, and genomic association with disease progression. Novel genetic findings may provide valuable information about this entity, which could open perspectives regarding novel therapeutic approaches for children with BCP-ALL iAMP21.