A positional scanning cyclic peptide library was generated using a penta-peptide thioester scaffold. Glycine was fixed at position R(1). Diaminopropionic acid was fixed at position R(3), with its γ-amino attaching to an anthraniloyl group. Positions R(2) and R(4) contained 36 L- and D- amino acids and position R(5) contained 19 L- amino acids. Cyclization was performed in a mixture of acetonitrile and 1.5 M aqueous imidazole solution (7:1 v/v) at room temperature for 5 days. No significant cross-oligomerization was detected under the cyclization conditions. The library was screened in a binding assay for mu opioid receptor, identifying the active amino acid mixture at each position. A total of 40 individual cyclic peptides were identified and synthesized by the combinations of the most active amino acid mixtures found at three positions 5 × 4 × 2. Two cyclic peptides exhibited high binding affinities to opioid receptor. The most active cyclic peptide in the library was yielded to have Tyr at R(2), D-Lys at R(4), and Tyr at R(5). Further investigation on this compound revealed the side chain-to-tail isomer to have greater binding affinity (14 nM) than the head-to-tail isomer (39 nM). Both isomers were selective for the mu-opioid receptor.