Background: Patients with pathological T1 (pT1) colorectal cancer (CRC) at high risk for lymph node metastasis (LNM) after complete local resection are recommended to receive additional intestinal resection along with lymph node dissection. However, the existing pathological criteria for risk stratification of LNM are inadequate, such that ~90% of patients without LNM are exposed to potentially unnecessary treatment. Based on the CIRCULATE-Japan platform, we launched DENEB, a new prospective substudy within the GALAXY observational study, to explore the ability of predicting LNM using circulating tumor DNA (ctDNA) detection compared to the standard pathological criteria. Methods: This study included patients with pT1 CRC who underwent complete local resection and were scheduled for additional intestinal resection with lymph node dissection based on the standard pathologic risk-stratification criteria for LNM. The additional surgery was indicated for patients meeting any of the following criteria: (1) depth of submucosal invasion (>1000µm); (2) lymphovascular invasion; (3) poorly differentiated adenocarcinoma, signet-ring cell carcinoma, or mucinous carcinoma; and (4) high-grade tumor budding (BD2/3) at the site of deepest invasion according to the Japanese guideline. ctDNA was analyzed from plasma samples collected within 4 weeks before the additional intestinal resection using a personalized, tumor-informed assay (Signatera bespoke multiplex-PCR NGS assay). The study assessed the diagnostic concordance rate between ctDNA detection and the occurrence of LNM. Results: Of 208 CRC patients enrolled between July 2021 and May 2023 in DENEB, 166 patients met the inclusion criteria. Of these, 22 (13.3%) were ultimately diagnosed with pStage III due to the presence of LNM in additional intestinal resection. Among the 166 patients, 6 patients tested ctDNA-positive; all 6 had LNM, yielding a positive predictive value of 100.0%. On the other hand, 160 patients had ctDNA-negative results, 144 of whom did not have LNM, resulting in a negative predictive value of 90.0%. The overall diagnostic concordance rate for ctDNA in detecting LNM was 90.4% (95% confidence interval, 84.8% to 94.4%). All 6 patients with a ctDNA-positive result and LNM had left-sided colorectal cancer and a tumor diameter greater than 13 mm. Conclusions: This analysis showed that ctDNA testing has the potential to improve risk stratification of LNM in patients with pT1 CRC who underwent complete local resection. Clinical trial information: UMIN000039205.