Introduction and Aim: Genetic and epigenetic factors affect chronic myeloid leukemia (CML) treatment response. This study aimed to investigate HOXA5 methylation and gene expression with Imatinib mesylate (IM) response in CML patients and assess their predictive value as response markers. Materials and Methods: Blood samples were collected from 50 CML patients (25 responders and 25 non-responders to Imatinib mesylate) and 50 healthy controls of the same age and sex. Methylation-specific quantitative PCR (MS-qPCR) was used to analyze HOXA5 methylation, and quantitative PCR was used to study gene expression (qPCR). Results: This study revealed a significant higher level of HOXA5 gene demethylation and expression in CML patients compared to control (P <0.001). Non-responder CML patients had significantly higher levels of HOXA5 gene demethylation and expression than responder CML patients (P<0.001 and P<0.01 respectively). According to the optimal cut-off point determined by receiver operating characteristics (ROC) analysis, a significant associated risk was observed between HOXA5 demethylation and expression levels (P<0.01 and P<0.01, respectively) and the development of IM resistance in CML patients. Conclusion: HOXA5 gene demethylation can be used as a biomarker to predict IM resistance in CML patients.