The thymus is the principal organ for development of T-cells. Thymocyte precursors from bone marrow-derived progenitor cells enter the thymus where they differentiate involving several differentiation stages into mature T-cells that can leave the thymus to the periphery. Migration of thymocytes through the thymus and their development are tightly controlled by the interaction of thymocytes with components of the thymic microenvironments. Several studies have demonstrated the pivotal importance of glycosylation in cell-cell interactions or interactions of cells with extracellular matrix components (ECM) in various physiologic processes in the body. The knowledge on glycosylation of thymic microenvironments is however limited although the presence of C-type lectin receptors such as DC-SIGN, mannose receptor and DEC-205, which are specifically recognizing distinct carbohydrate moieties emphasize the importance of glycosylation in the thymus. In order to outline the distribution of glycoconjugates in microenvironments of the human thymus we studied the glycosylation of the human thymic microarchitecture by using plant lectins in situ. Eleven plant lectin-biotin conjugates with distinct specificity were used and analyzed by fluorescence microscopy. Mannose glycoconjugates, specifically detected by the lectins GNA and NPA, were abundant in the cortex but not in the medulla. Dendritic cells present in the thymic cortex were specifically co-stained with the galactose-specific lectins DSA and PNA. Several lectins bound to the thymic vasculature. The alpha2-fucose-specific lectin UEA stained thymic blood vessels in the interlobular space and medulla and capillaries in the cortex. In addition to UEA, thymic blood vessels and capillaries also reacted with the lectins DSA, PNA and the alpha-GalNac-specific lectin HPA. In contrast, lymph vessels present in the interlobular space do not interact with UEA, DSA and PNA, but only with HPA, revealing a disparate glycosylation pattern of lymph and blood vessels that may be important to determine the direction of thymocytes entering or leaving the thymus. In conclusion, the restricted expression patterns of carbohydrates defined microenvironments in the human thymus highlight the importance of glycosylation in various steps of T-cell development.