Glycan microarrays are an increasingly utilised tool for analysis of protein-carbohydrate interactions and a variety of glycan-containing molecules and slide chemistries have been used to array carbohydrates on microarray surfaces. Slide surface chemistry can have significant impact on the ligand presentation, background noise, spot size and morphology and reproducibility of the arrayed molecules, which in turn impacts upon lectin-carbohydrate recognition. The linker used to attach the carbohydrate to the molecular scaffold is another variable in ligand presentation. To evaluate these effects, three different microarray surface chemistries were arrayed with the same mono-and disaccharide neoglycoconjugates and natural glycoproteins and incubated with four well-characterised plant lectins. Analogues of three monosaccharide neoglycoconjugates, with two common linkers each, were included in the test group to evaluate the linker effect on lectin recognition. Based on lowest background noise, expected lectin-ligand interaction, good spot morphology and best reproducibility, the three-dimensional hydrogel slide surface proved most suitable for lectin interrogation of carbohydrate ligands, and the more flexible phenylisothiocyanate linker afforded greater recognition of the carbohydrates by the relevant lectins.