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Background: Chronic myeloid leukemia (CML) is a hematopoietic stem cell (SC) neoplasm diagnosed by the demonstration of t(9;22)(BCR‐ABL1) fusion gene. We performed a flow cytometric assay to identify CD26+ CML leukemic stem cells (LSCs) for its value as a standalone diagnostic investigation for CML and its utility for detection of residual disease in CML patients on therapy. Methods: Patients with clinical suspicion of CML/CML on follow‐up were included, and peripheral (PB) and/or bone marrow (BM) samples were utilized for flow cytometric analysis. PB and/or BM of patients with diseases other than CML were used as controls. A pre‐titrated antibody cocktail containing CD45, CD34, CD38, and CD26 MoABs was used. Results: A total of 104 samples (63 PB and 41 BM) from 64 patients [suspected CML (n = 30), CML on follow‐up (n = 15), and non‐CML (n = 19)] were tested. CD26+ LSCs were identified in all patients with a confirmed diagnosis of CML (median = 0.07 (range 0.002%–26.79%)). None of the patients in the control group (non‐CML) and follow‐up patients with negative reverse transcriptase‐polymerase chain reaction (RT‐PCR) results showed the presence of CD26+ LSCs. Also, there was a strong correlation between CD26+ CML LSCs in the PB and BM (r =.917). Conclusion: Flow cytometric identification of CD26+ LSCs in the peripheral blood can be a cheap, rapid, robust, and potential diagnostic tool for the diagnosis of CML compared to available testing methods. It is irrespective of BCR‐ABL1 transcript type, and its role in residual disease monitoring needs thorough investigation. [ABSTRACT FROM AUTHOR]