A glycopeptide library containing more than 500,000 compounds has been constructed from a combination of Asn-linked carbohydrates using one-bead-one-compound combinatorial methodologies. The library was encoded with peptide markers that were topologically segregated on the interior of the solid support to negate interference with carbohydrate/protein recognition during lectin screening. Both the peptide backbone and carbohydrate components were randomized, but the glycosamine was limited to position 3 at the center of the pentapeptide to evaluate the influence of the peptide backbone in lectin recognition. Of the four lectins that were evaluated, remarkable selectivity was observed with wheat germ agglutinin (WGA), which recognizes N-acetyl glucosamine (GlcNAc). From more than 80,000 possible combinations, only six ligands were identified, all possessing GlcNAc. These compounds were independently synthesized, characterized, and evaluated in solution. All six of the glycopeptides showed higher affinity for WGA than GlcNAc, with one having a 4-fold increase. Modeling studies indicate that the peptide backbone is capable of interacting with amino acids in the active site of WGA, but these interactions are not strongly correlated with activity, suggesting that the primary role of the peptide is to properly orient the sugar in the recognition process.