Abstract Background Aberrant DNA methylation plays a crucial role in the progression of myeloid neoplasms. Previously, our literature reported that slit guidance ligand 2 (SLIT2) promoter methylation was associated with disease progression and indicated a poor prognosis in patients with myelodysplastic syndrome. Herein, we further investigated the clinical implications and role of SLIT2 promoter methylation in patients with chronic myeloid leukemia (CML). Methods The level of SLIT2 promoter methylation was determined in 104 CML patients, and its clinical significance was analyzed. Moreover, demethylation studies were performed in K562 cells to determine the epigenetic mechanism by which SLIT2 promoter methylation is regulated in CML. Results The level of SLIT2 promoter methylation was similar between CML patients and controls. However, deeper analysis revealed that the SLIT2 promoter methylation level in the accelerated phase (AP) and blast crisis (BC) was markedly higher than that in the chronic phase (CP) and controls. Additionally, a marked difference was identified between the SLIT2 promoter hypermethylated and non-hypermethylated groups among CML patients grouped by clinical stage. The frequency of SLIT2 hypermethylation was markedly increased with the progression of clinical stage, that is, it was the lowest in CP samples (12/80, 15%), higher in AP samples (4/8, 50%) and the highest in BC samples (11/16, 69%). Importantly, the level/density of SLIT2 promoter methylation was significantly higher in the advanced stage than in the early stage among the 6 tested paired CML patients. Epigenetically, the expression of the SLIT2-embedded non-coding genes SLIT2-IT1 and miR-218 expression was decreased in patients with CML. SLIT2 promoter hypermethylated cases had a markedly lower SLIT2-IT1 expression level than SLIT2 promoter non-hypermethylated cases. Moreover, SLIT2-IT1 and miR-218 expression was remarkably upregulated in a dose-dependent manner after demethylation treatment of K562 cells. Conclusions Hypermethylation of the SLIT2 promoter is correlated with disease progression in CML. Furthermore, SLIT2 promoter methylation may function by regulating the expression of the SLIT2-embedded non-coding genes SLIT2-IT1 and miR-218 during CML progression.