Targeted molecular imaging may overcome current challenges in the preoperative and intraoperative delineation of pancreatic ductal adenocarcinoma (PDAC). Tumor-associated glycans Lea/c/x, sdi-Lea, sLea, sLex, sTn as well as mucin-1 (MUC1) and mucin-5AC (MU5AC) have gained significant interest as targets for PDAC imaging. To evaluate their PDAC molecular imaging potential, biomarker expression was determined using immunohistochemistry on PDAC, (surrounding) chronic pancreatitis (CP), healthy pancreatic, duodenum, positive (LN+) and negative lymph node (LN−) tissues, and quantified using a semi-automated digital image analysis workflow. Positive expression on PDAC tissues was found on 83% for Lea/c/x, 94% for sdi-Lea, 98% for sLea, 90% for sLex, 88% for sTn, 96% for MUC1 and 67% for MUC5AC, where all were not affected by the application of neoadjuvant therapy. Compared to PDAC, all biomarkers were significantly lower expressed on CP, healthy pancreatic and duodenal tissues, except for sTn and MUC1, which showed a strong expression on duodenum (sTn tumor:duodenum ratio: 0.6, p < 0.0001) and healthy pancreatic tissues (MUC1 tumor:pancreas ratio: 1.0, p > 0.9999), respectively. All biomarkers are suitable targets for correct identification of LN+, as well as the distinction of LN+ from LN− tissues. To conclude, this study paves the way for the development and evaluation of Lea/c/x-, sdi-Lea-, sLea-, sLex- and MUC5AC-specific tracers for molecular imaging of PDAC imaging and their subsequent introduction into the clinic.