High hyperdiploid (HeH) B-cell acute lymphoblastic leukemia (B-ALL) is the most prevalent subtype of childhood ALL. This leukemia is characterized by trisomies and tetrasomies of specific chromosomes and additional point mutations. Here, we used single-cell targeted DNA and antibody sequencing to determine the clonal evolution of HeH B-ALL during development and chemotherapy treatment. Chromosomal copy number changes were mostly stable over all the leukemia cells, while mutations were typically subclonal. Within all 13 cases, at least one mutant ( or ) subclone was detected (range: 1 to 4 subclones with mutations), indicating the importance of signaling in HeH B-ALL development. mutations were detected in 4 out of 13 cases and always in a subclone with signaling mutations. Single-cell DNA sequencing detected residual leukemia cells during chemotherapy treatment, and analysis of chromosomal copy number changes aided in the accurate detection of these cells. Our single-cell data demonstrate that chromosomal changes are acquired prior to additional mutations and that RAS signaling mutations are present in all HeH cases, often as subclonal mutations. This single-cell multi-omics study enabled us to extensively characterize the genetic and surface protein heterogeneity in patients with HeH B-ALL.