Chronic myeloid leukemia (CML) is genetically characterized by the Philadelphia (Ph) chromosome, formed through a reciprocal translocation between chromosomes 9 and 22 and giving rise to the constitutively active tyrosine kinase P210 BCR/ABL1. Therapeutic strategies aiming for a cure of CML will require full eradication of Ph chromosome-positive (Ph⁺) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34⁺ cells and also in cord blood CD34⁺ cells as a consequence of retroviral BCR/ABL1 expression. To test whether IL1RAP expression distinguishes normal (Ph⁻) and leukemic (Ph⁺) cells within the CML CD34⁺CD38⁻ cell compartment, we established a unique protocol for conducting FISH on small numbers of sorted cells. By using this method, we sorted cells directly into drops on slides to investigate their Ph-chromosome status. Interestingly, we found that the CML CD34⁺CD38⁻IL1RAP⁺ cells were Ph⁺, whereas CML CD34⁺CD38⁻IL1RAP⁻ cells were almost exclusively Ph⁻. By performing long-term culture-initiating cell assays on the two cell populations, we found that Ph⁺ and Ph⁻ candidate CML stem cells could be prospectively separated. In addition, by generating an anti-IL1RAP antibody, we provide proof of concept that IL1RAP can be used as a target on CML CD34⁺CD38⁻ cells to induce antibody-dependent cell-mediated cytotoxicity. This study thus identifies IL1RAP as a unique cell surface biomarker distinguishing Ph⁺ from Ph⁻ candidate CML stem cells and opens up a previously unexplored avenue for therapy of CML.