Specific targeting of the integrin subtype α5β1 possesses high potential in cancer diagnosis and therapy. Through sequential N-methylation, we successfully converted the biselective α5β1/αvβ6 peptide c(phg- isoDGR-k) into a potent peptidic RGD binding α5β1 subtype selective ligand c(phg- isoDGR-( NMe)k). Nuclear magnetic resonance spectroscopy and molecular modeling clarified the molecular basis of its improved selectivity profile. To demonstrate its potential in vivo, c(phg- isoDGR-( NMe)k) was trimerized with the chelator TRAP and used as a positron-emission tomography tracer for monitoring α5β1 integrin expression in a M21 mouse xenograft.