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