Background: Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by proliferation of immature myeloid cells maintaining their capacity to differentiate. The increase of myeloid precursors is due to an acquired genetic alteration of the hematopoetic stem cells that behave as leukemic stem cells (LSC). It is characterized by the chromosomal translocation t(9;22)(q34.1;q11.2), which results in the formation of the Philadelphia chromosome, containing the BCR-ABL1 fusion gene. The diagnosis must be confirmed by cytogenetic analysis and RT-qPCR. In 2019, Raspadori et al. described a flow cytometry protocol for CML diagnosis and identified the expression of CD26 as a marker for CML LSC in peripheral blood and bone marrow. Aim: To compare a flow cytometry protocol for CML investigation with the gold standard diagnostic method BCR-ABL PCR assay. Methods: Peripheral blood and bone marrow samples received at Sabin Medicina Diagnostica lab between April 2019 and January 2020 for CML investigation with medical order for flow cytometry and PCR for BCR-ABL assays were retrospectively analyzed. For flow cytometry, samples were processed according to protocol previously described by Raspadori et al. and stained with the following anti-human monoclonal antibodies: HLA-DR-FITC (G46-6), CD123-PE (9F5), CD34-PerCP-Cy5.5 (8G12), CD117PE-Cy7 (104D2), CD38-APC-H7(HB7), CD33 BV421 (WM53), CD45-V500 (2D1) from BD Biosciences and CD26-APC (BA5b) from Exbio. Acquisition was performed on a 3-lasers, 8-colors FACSCanto™II flow cytometer (BD Bioscence). Analysis and quantification of CML LSC (CD34+/CD26+/CD38-) was performed using Infinicyt (Cytognos). BCR-ABL1 was identified by an in house routine one-step RT-qPCR using ΔΔCq method. The buffy coats were removed from EDTA-whole blood (8 mL) or bone marrow samples (4mL), nucleic acids were stabilized and then extracted using Magna 96 (Roche). A one-step RT-qPCR QuantiNova Probe master mix (Qiagen) with primers and probes described by Gabert et al. 2003 (EAC) and by Pane et al. 1996 were used. BCR-ABL1 and ABL RNAs were co-amplified at Roche LightCycler 480II for e14a2/e13a2 (p210), e1a2 (p190), and e19a2 (p230) fusions. The degree of agreement between the test methods (flow cytometry) and the comparative methods (PCR for BCR-ABL) was quantified using Kappa statistics with three categories. Results: In this period, 21 samples from different patients were received at Sabin Medicina Diagnostica lab for CML investigation and medical order for flow cytometry and BCR-ABL assays. 10 samples were from peripheral blood and 11 from bone marrow. In 10 samples (3 bone marrows and 7 peripheral blood), flow cytometry assay did not show a CD26+ CML LSC population, and BCR/ABL PCR assay resulted negative. In 11 samples (8 bone marrows and 3 peripheral blood), a CD26+ CML LSC population was identified by flow cytometry and BCR-ABL PCR assay resulted positive. There were no discordant results. The degree of agreement between the test methods (flow cytometry) and the comparative methods (PCR for BCR-ABL) was a perfect agreement (kappa=1). Conclusions: In conclusion, our data are in accordance with the results previously described by Raspadori et al. Although we still believe that further studies are necessary, the identification of a CD26+ CML LSC population by flow cytometry may be a diagnostic tool for CML when a BCR-ABL PCR assay is not available.