Epigenetic analysis of the association between the methylation status of the promoter region of the MTRR (5‑methyltetrahydrofolate‑homocysteine methyltransferase reductase) gene and the risk of acute lymphoblastic leukemia (ALL) in children plays an important role in the early diagnosis, assessment of the malignant degree, treatment and evaluation of the risk of relapse and prognosis of the disease. In the present study, RT‑qPCR was used to detect the mRNA levels of the MTRR and MTHFR (methylenetetrahydrofolate reductase) genes in the bone marrow of 20 ALL patients and 20 age‑ and sex‑matched controls with normal bone marrow. The methylation pattern of the MTRR promoter region in eligible DNA samples was quantitatively analyzed using MALDI‑TOF MS. The results indicated that the mRNA expression level of MTRR in the bone marrow from children with ALL was lower than that in the control samples (P<0.05), but no significant difference was detected in the MTHFR gene between the two groups (P>0.05). According to the risk classification of ALL in children with high, medium and low risk, the low‑risk group had a higher methylation rate of CpG_6 compared to the medium‑risk group. However, the medium‑risk group had a higher CpG_46.47 methylation rate compared to the low‑risk group. The methylation rates of CpG_26 and CpG_46.47 in the high‑risk group were higher than these rates in the low‑risk group, while the CpG_42.23.44 methylation rate was lower in the high‑risk group than in the low‑risk group (P<0.05). The methylation rates at CpG_1, CpG_10, CpG_48 sites, score and the average methylation rate in the ALL‑H (high) group (≥50x109/l) were lower than these in the ALL‑NH (not high) group (<50x109/l) and the control group (P<0.05). We conclude that abnormal MTRR mRNA expression and the methylation of the MTRR promoter can be used to classify the risk of ALL in children.