Lymph node metastasis (LNM) is the most important prognostic factor in patients undergoing surgical resection of pancreatic ductal adenocarcinoma (PDAC). In this study, we aimed to identify molecular markers associated with LNM in PDAC using genome-wide expression profiling. In this study, laser microdissection and genome-wide transcriptional profiling were used to identify genes that were differentially expressed between PDAC cells with and without LNM obtained from 20 patients with PDAC. Immunohistochemical staining was used to confirm the clinical significance of these markers in an additional validation set of 43 patients. In the results, microarray profiling identified 46 genes that were differently expressed between PDAC with and without LNM with certain significance. Four of these biomarkers were validated by immunohistochemical staining for association with LNM in PDAC in an additional validation set of patients. In 63 patients with PDAC, significant LNM predictors in PDAC elucidated from multivariate analysis were low expression of activating enhancer binding protein 2 (AP2α) (P = 0.012) and high expression of mucin 17 (MUC17) (P = 0.0192). Furthermore, multivariate analysis revealed that AP2α-low expression and MUC17-high expression are independent prognostic factors for poor overall survival (P = 0.0012, 0.0001, respectively). In conclusion, AP2α and MUC17 were independent markers associated with LNM of PDAC. These two markers were also associated with survival in patients with resected PDAC. We demonstrate that AP2α and MUC17 may serve as potential prognostic molecular markers for LNM in patients with PDAC.