Simple SummaryLeukemia is the most common type of childhood malignancy. While the clinical management of pediatric acute leukemia, especially acute lymphoblastic leukemia, underwent a remarkable improvement during the past decades, a subset of the patients still experience relapse and succumb to their disease. Emergence or repositioning of targeted therapies aided by the comprehensive characterization of single leukemic cells using advanced sequencing approaches may provide novel opportunities for therapeutic intervention in these patients. In our review, we summarize the status quo of single-cell-sequencing studies in pediatric leukemia, provide an overview of the current landscape of targeted agents applicable in this disease group, and highlight options for ways single-cell sequencing could further support the decision making related to the clinical management of pediatric leukemia.Single-cell sequencing (SCS) provides high-resolution insight into the genomic, epigenomic, and transcriptomic landscape of oncohematological malignancies including pediatric leukemia, the most common type of childhood cancer. Besides broadening our biological understanding of cellular heterogeneity, sub-clonal architecture, and regulatory network of tumor cell populations, SCS can offer clinically relevant, detailed characterization of distinct compartments affected by leukemia and identify therapeutically exploitable vulnerabilities. In this review, we provide an overview of SCS studies focused on the high-resolution genomic and transcriptomic scrutiny of pediatric leukemia. Our aim is to investigate and summarize how different layers of single-cell omics approaches can expectedly support clinical decision making in the future. Although the clinical management of pediatric leukemia underwent a spectacular improvement during the past decades, resistant disease is a major cause of therapy failure. Currently, only a small proportion of childhood leukemia patients benefit from genomics-driven therapy, as 15–20% of them meet the indication criteria of on-label targeted agents, and their overall response rate falls in a relatively wide range (40–85%). The in-depth scrutiny of various cell populations influencing the development, progression, and treatment resistance of different disease subtypes can potentially uncover a wider range of driver mechanisms for innovative therapeutic interventions.