Abstract Background: Ampulla of Vater (AoV) carcinoma is a rare tumor that accounts for 20 % of periampullary cancers. Surgical resection is the only treatment that can expect cure of disease, and there is still an unmet need to predict postoperative recurrence. In this study, we investigated the novel molecular biomarkers that can predict the risk of recurrence after surgery with AoV carcinoma. Methods: In this retrospective study, we collected the surgically resected tissue and medical records of patients who were diagnosed with AoV carcinoma at Samsung Medical Center from January 2005 to December 2020. Patients were classified patients according to the initial stage and experience of tumor recurrence. Whole transcriptomic analysis using primary AOV tumor and recurred tumor tissue samples was conducted to identify the molecular biomarker for tumor recurrence. Results: A total of 40 tumor tissue from primary site in non-recurrence group, 22 tumor tissue from primary site in recurrence group, and paired 22 tumor tissue from recurrence site were analyzed. A total of 1005 genes differentially expressed between primary tumor of non-recurrence group and recurrence group were found. Gene set enrichment analysis showed that pathways related to fatty acid metabolism, adipogenesis, oxidative phosphorylation, and hypoxia were upregulated, and pathways associated with interferon gamma response, and mTOR signaling were downregulated in recurrence group. These pathways were significantly affecting the recurrence free survival which was verified with TCGA data, and we further identified some candidate genes including PIM3, IGFBP4, SLC19A1, HIF1A and CXCL11 to predict tumor recurrence . Moreover, we found candidates for single gene level biomarkers to predict early tumor recurrence in N0 stage. Low expression of UQCC3 and high expression of CLCNKB was related to early tumor recurrence. Conclusions: We found the candidates for novel molecular level biomarker to predict postoperative recurrence of AoV carcinoma. In the future, it is necessary to develop a predictive model for the risk of recurrence after surgical resection for AoV carcinoma. Citation Format: Hyemin Kim, Jin Ho Choi, Hyoung-oh Jeong, Aryoung Kim, Se-Hoon Lee, Kwang Hyuck Lee, Kyu Taek Lee, Jong Kyun Lee, Semin Lee, Joo Kyung Park. Identification of novel molecular biomarkers for predicting postoperative recurrence of ampulla of Vater carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5197.