Juvenile Myelomonocytic Leukemia (JMML) is a poor prognosis childhood leukemia usually caused by germline or somatic RAS-activating mutations. The cellular hierarchy in JMML is poorly characterized, including the identity of leukemia stem cells (LSCs). FACS and single-cell RNA-sequencing reveal marked heterogeneity of JMML hematopoietic stem/progenitor cells (HSPCs), including an aberrant Lin-CD34+CD38-CD90+CD45RA+ population. Single-cell HSPC index-sorting and clonogenic assays show that (1) all somatic mutations can be backtracked to the phenotypic HSC compartment with RAS -activating mutations as a “first hit”, (2) mutations are acquired with both linear and branching patterns of clonal evolution and (3) mutant HSPCs are present after allogeneic HSC transplant before molecular/clinical evidence of relapse. Stem cell assays reveal inter-patient heterogeneity of JMML-LSCs which are present in, but not confined to, the phenotypic HSC compartment. RNA-sequencing of JMML-LSCs reveals upregulation of stem cell and fetal genes ( HLF, MEIS1, CNN3 , VNN2 , HMGA2 ) and candidate therapeutic targets/biomarkers ( MTOR, SLC2A1 , CD96 ) paving the way for LSC-directed disease monitoring and therapy in this disease.