Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N-glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor-specific N-glycan alterations in ovarian cancer development and progression. matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N-glycan distribution on formalin-fixed paraffin-embedded ovarian cancer tissue sections from early- and late-stage patients. Tumor-specific N-glycans are identified and structurally characterized by porous graphitized carbon-liquid chromatography-electrospray ionization-tandem mass spectrometry (PGC-LC-ESI-MS/MS), and then assigned to high-resolution images obtained from MALDI-MSI. Spatial distribution of 14 N-glycans is obtained by MALDI-MSI and 42 N-glycans (including structural and compositional isomers) identified and structurally characterized by LC-MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N-glycan families are localized to the tumor regions of late-stage ovarian cancer patients relative to early-stage patients. Potential N-glycan diagnostic markers that emerge include the oligomannose structure, (Hex)6 + (Man)3 (GlcNAc)2 , and the complex neutral structure, (Hex)2 (HexNAc)2 (Deoxyhexose)1 + (Man)3 (GlcNAc)2 . The distribution of these markers is evaluated using a tissue microarray of early- and late-stage patients.