We describe the formation of self-assembled monolayers (SAMs) on gold by helical β-peptides and show that β-peptide sequence can determine whether or not the SAM displays internal order. β-Peptide sequence isomers were designed either to be globally amphiphilic (GA), i.e., to display segregated hydrophilic and lipophilic surface patches, or to display an even distribution of hydrophilic and lipophilic side chains (iso-GA). These isomers were compared in terms of two-dimensional self-assembly on a gold surface. β-Peptides derivatized with 3-mercapto-propionic acid, termed GA-SH and iso-GA-SH, display an N-terminal thiol for formation of gold-sulfur bonds. Comparison of ellipsometry measurements and the Amide I /Amide II ratios from infrared spectroscopy indicates that interactions between globally amphiphilic β-peptide molecules dictate the organization of the SAM formed from GA-SH. In contrast, the non-globally amphiphilic isomer iso-GA-SH formed a disordered monolayer. Comparison of the IR data for GA-SH and an analogue lacking a terminal thiol group, GA-NH2, suggests that thiol-mediated chemisorption to the gold surface is not required for organization but facilitates the concentration of β-peptides at the surface. These studies suggest that sequence control among β-peptides is important for generating an ordered monolayer. Because the helices formed by appropriately designed β-peptides are more stable than helices formed by α-peptides, we suggest that β-peptide SAMs provide an avenue to the rational engineering of surfaces in which chemical groups are presented in precise and predictable arrangements.