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Background The P‐glycoprotein (P‐gp) is one of the mechanisms of Imatinib (IM) resistance in chronic myeloid leukemia (CML). P‐gp has been identified as an efflux pump involved in releasing of IM outside CML cells. To date, the P‐gp involvement in the IM resistance development was not completely understood. Therefore, the present study aimed at measuring the P‐gp expression level on lymphocytes from Tunisian patients with CML and correlating this level with a molecular response to IM. Method The expression of P‐gp on peripheral blood lymphocytes from 59 Tunisian patients with CML (27 IM responder patients vs 32 IM non‐responder patients) was evaluated by flow cytometry. Result Our finding showed significantly positive expression of P‐gp in the lymphocytes from the IM non‐responder group when compared to the IM‐responder group (P = .001). In IM non‐responder CML patients, the comparison between CCyR achievers and non‐achievers showed a high mean fluorescence intensity (MFI) of P‐gp expression in patients who did not achieve their CCyR (P = .001). The comparison between patients with primary and secondary resistance to IM showed an increasing MFI value in patients with primary resistance to IM (P = .001). Besides, the comparison between nilotinib‐treated and dasatinib‐treated patients proved a high value of MFI in nilotinib‐treated patients (P = .001). Conclusion The overexpression of P‐gp on lymphocytes has significantly correlated with the failed molecular response to IM in patients with CML.