We tested the substrate range of four wild-type Escherichia coli aminoacyl-tRNA synthetases (AARSs) with a library of non-standard amino acids (nsAAs). While these AARSs could discriminate efficiently against the other canonical amino acids, they were able to use many nsAAs as substrates. Our results also showed E. coli tryptophanyl-tRNA synthetase (TrpRS) and tyrosyl-tRNA synthetase to have overlapping substrate ranges. In addition, we found that the nature of the anticodon sequence of tRNATrp altered the nsAA substrate range of TrpRS; this implies that the sequence of the anticodon affects the TrpRS amino acid binding pocket. These results highlight again that inherent AARS polyspecificity will be a major challenge to the goal of incorporating multiple different amino acids site-specifically into proteins.