Imatinib introduction caused to improve the clinical outcomes of chronic myeloid leukemia (CML) patients. Despite the significant effects of Imatinib, pharmacogenetic variables induced treatment resistant is also observed. Imatinib is known as a P-glycoprotein (P-gp) efflux pump substrate encoded by the ABCB1 gene. The ABCB1 C1236T, G2677T/A and C3435T variants are possibly correlated with interindividual variation in pharmacokinetic response to Imatinib therapy. The present study aimed to examine the effect of ABCB1 gene variants on the therapeutic response of Imatinib in CML patients. Sixty-nine Iranian CML patients treated with Imatinib or Nilotinib were selected and divided into two groups of sensitive and resistant to Imatinib. C1236T and G2677T/A variants were genotyped by high resolution melting (HRM) analysis, and C3435T variant was genotyped using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). Then, the results were compared between the two groups of patients. Our results showed that there were no significant differences between C1236T, G2677T/A and C3435T variants of ABCB1 gene and clinical response to Imatinib in the Iranian CML patients. According to the results of this study, genotyping of ABCB1 C1236T, G2677T/A and C3435T variants couldn’t help to predict the outcomes of Imatinib treatment in CML patients. So, these variants are not useful to make decisions about treatment, but it is suggested to do further investigations.