Background/Objectives: Intrachromosomal amplification of chromosome 21 (iAMP21) represents a rare and heterogeneous distinct cytogenetic subgroup of B-cell precursor acute lymphoblastic leukemia (ALL) initially associated with a poor prognosis. Treatment intensification with additional doses of methotrexate and asparaginase was associated with better treatment outcomes. Methods: In this retrospective single-center study, we evaluated the impact of iAMP21 on treatment outcome in a cohort of pediatric patients treated with an intensified asparaginase regimen and describe the genomic landscape of four patients with iAMP21. Results: Four out of 89 patients > 1 year old were classified as iAMP21 positive. Five-year event-free survival (EFS) was inferior in the iAMP21-positive group: 25% versus 85.6% (p = 0.001). The cumulative incidence of relapse and treatment-related mortality were 50% vs. 9.9% and 0% vs. 2.38%, respectively, in the iAMP21-positive and non-iAMP21 groups (p = 0.02 and 0.76, respectively). These results did not translate into a significant difference in overall survival: 100% vs. 93.7% (p = 0.6). The presence of iAMP21 (HR 7.68, 95% CI 2.04–29.05; p = 0.002) and a measurable residual disease ≥1% after induction on day +33 (HR 8.82, 95% CI 2.6–29.91; p = 0.001) retained significant negative impact on EFS in multivariate analysis. Conclusions: We found an independent significant prognostic impact of iAMP21 on EFS among pediatric patients with ALL, and clinical presentation and early treatment response did not classify these patients as HR. Diverse genetic backgrounds among iAMP21-positive patients might influence the treatment response and outcome of this heterogeneous disease.