The current surge in defining glycobiomarkers by applying lectins rekindles interest in definition of the sugar-binding sites of lectins at high resolution. Natural complex-type N-glycans can present more than one potential binding motif; posing the question of the actual mode of interaction when interpreting, for example, lectin array data. By strategically combining N-glycan preparation with saturation-transfer difference NMR and modeling, we illustrate that epitope recognition depends on the structural context of both the sugar and the lectin (here, wheat germ agglutinin and a single hevein domain) and cannot always be predicted from simplified model systems studied in the solid state. We also monitor branch-end substitutions by this strategy and describe a three-dimensional structure that accounts for the accommodation of the alpha 2,6-sialylated terminus of a biantennary N-glycan by viscumin. In addition, we provide a structural explanation for the role of terminal alpha 2,6-sialylation in precluding the interaction of natural N-glycans with lectin from Maackia amurensis. The approach described is thus capable of pinpointing lectin-binding motifs in natural N-glycans and providing detailed structural explanations for lectin selectivity.