This study aimed to unravel the lymph node metastasis (LNM)-related methylated DNA (mDNA) landscape and develop a mDNA signature to identify LNM in patients with T1 colorectal cancers (T1 CRC).Considering the invasiveness of T1 CRC, current guidelines recommend endoscopic resection in patients with LNM-negative, and radical surgical resection only for high-risk LNM-positive patients. Unfortunately, the clinicopathological criteria for LNM risk-stratification are imperfect, resulting in frequent misdiagnosis leading to unnecessary radical surgeries and post-surgical complications.We conducted genome-wide methylation profiling of 39 T1 CRC specimens to identify differentially methylated CpGs between LNM-positive and LNM-negative, and performed quantitative pyrosequencing analysis in 235 specimens from three independent patient cohorts, including 195 resected tissues (training cohort: n=128, validation cohort: n=67) and 40 pre-treatment biopsies.Using logistic regression analysis, we developed a nine-CpG signature to distinguish LNM-positive versus LNM-negative surgical specimens in the training cohort (area under the curve [AUC]=0.831, 95% confidence interval [CI]=0.755-0.892; P0.0001), which was subsequently validated in additional surgical specimens (AUC=0.825; 95% CI=0.696-0.955; P=0.003) and pre-treatment biopsies (AUC=0.836; 95% CI=0.640-1.000, P=0.0036). This diagnostic power was further improved by combining the signature with conventional clinicopathological features.We established a novel epigenetic signature that can robustly identify LNM in surgical specimens and even pre-treatment biopsies from patients with T1 CRC. Our signature has strong translational potential to improve the selection of high-risk patients who require radical surgery while sparing others from its complications and expense.