Using a recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method, we have spatially profiled N-linked glycans in human breast cancer formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMA). Routinely, 50 or more individual N-glycan species are detected per FFPE tissue, and this number increases significantly with disease severity. Through the combined analysis of breast cancer FFPE tissues and TMAs we have been able to identify histopathology co-localized glycan structural classes specific to stromal, necrotic and tumor regions. N-glycan panels derived from TMAs representing HER2 receptor positive and triple negative breast cancers were compared to identify shared and divergent glycosylation patterns between the two genetic sub-types. Within the tissues, we have identified a series of high-mannose glycans that are predominantly associated with tumor regions, as well as more highly branched and fucosylated glycans in higher grade tumors. In addition to this, we also found a series of polylactosamine glycans with increasing expression in HER2+, triple negative and metastatic breast cancers. The expression of these polylactosamine glycans could provide further mechanistic insights into the development and spread of breast cancer, and provide new research directions for identifying prognostic markers. This article is protected by copyright. All rights reserved.