Protein glycosylation is a posttranslational modification, which can result in functional changes of the glycoproteins and can play a key role in cancer progression and treatment. Here, we have analysed formalin fixed paraffin embedded (FFPE) endometrial cancer and adjacent tissues to explore tissue specific N-linked glycan abundance using glycan matrix assisted laser desorption ionisation mass spectrometry imaging (MALDI MSI). We have used single sections and tissue microarrays (TMA) in our analysis. Identification of discriminative m/z values was achieved using porous graphitized carbon liquid chromatography (PGC-LC) and collision induced electrospray negative mode MS fragmentation analysis (ESI-MS/MS). High mannose glycan structures were predominately detected in the tumour regions, while complex bi- and tri-antennary structures were observed in the normal/adjacent regions, indicating a biological role for these modifications. In summary, tumour and normal regions were clearly distinguished based on their N-glycan distributions.