Aim: In recent years, a few cases of chronic myeloid leukemia (CML) have been reported with both BCR-ABL and JAK2V617F mutations. Moreover, mutations in the additional sex comb-like 1 (ASXL1) gene were recently shown in various myeloid malignancies. There were no previous studies investigating the incidence of the ASXL1 and JAK2V617F mutations in Iranian patients with CML. Consequently, this study focuses on the analysis of these mutations in patients with CML. Methods: In total, 66 patients with a clinical diagnosis of CML were examined at the time of diagnosis. Thirty healthy subjects were checked as controls. Exon 12 of ASXL1 was amplified from genomic DNA and bidirectionally sequenced. We also performed JAK2V617F screening by amplification refractory mutation system-polymerase chain reaction and sequencing. Results: Mutations in the ASXL1 gene were found in five out of 66 CML patients (7.6%). We identified a novel variant (c.1968G >A, p.Asp656Asn) in one of the patients that has not been reported before. We also identified BCR-ABL and JAK2V617F mutations simultaneously in four patients (6%). Conclusion: Our demonstration of ASXL1 mutation, a putative tumor suppressor gene, represents an important molecular abnormality in CML. We also showed that concomitant detection of BCR-ABL and JAK2V617F mutations has a relatively high incidence in Iranian patients.