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Whole-genome bisulfite sequencing (WGBS) was performed on four monozygotic and one dizygotic twin pairs with discordant B-ALL status, providing a unique experimental model to dissect relapse-associated epigenetic alterations while controlling for genetic and environmental confounders. All affected twins achieved post-chemotherapy remission but later relapsed. Findings were validated in 149 ALL patients, 135 healthy siblings, and 1,443 public samples. Bioinformatics analysis identified differentially methylated regions (DMRs), significantly enriched in regulatory elements, and a 10-gene methylation panel was developed using a random forest algorithm