Pancreatic ductal adenocarcinoma (PDAC) tumor heterogeneity impedes the development of biomarker assays for early disease detection. We hypothesized that PDAC cell subpopulations could be identified by aberrant glycan signatures in both tumor tissue and blood samples. We used multiplexed glycan immunofluorescence to distinguish between PDAC and noncancer cell subpopulations within tumor tissue, and we developed hybrid glycan sandwich assays to determine whether the aberrant glycan signatures could be detected in blood samples. We found that PDAC cells were identified by signatures of glycans detected by four glycan-binding proteins (VVL, CA19-9, sTRA, and GM2) and that there are three types of glycan-defined PDAC tumors: sTRA type, CA19-9 type, and intermixed. In patient-matched tumor and blood samples, the PDAC tumor type could be determined by the aberrant glycans in the blood. As a result, the combined assays of aberrant glycan signatures were more sensitive and specific than any individual assay. Our results demonstrate a methodology to detect and stratify PDAC.