Genetically encoding distinct non-canonical amino acids (ncAAs) into proteins synthesized in cells requires mutually orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The pyrrolysyl-tRNA synthetase/tRNA pair from Methanosarcina mazei (Mm) has been engineered to incorporate diverse ncAAs and is commonly considered an ideal pair for genetic code expansion. However, finding new aaRS/tRNA pairs that share the advantages of the MmPylRS/MmtRNA pair and are orthogonal to both endogenous aaRS/tRNA pairs and the MmPylRS/MmtRNA pair has proved challenging. Here we demonstrate that several ΔNPylRS/tRNA pairs, in which PylRS lacks an N-terminal domain, are active, orthogonal and efficiently incorporate ncAAs in Escherichia coli. We create new PylRS/tRNA pairs that are mutually orthogonal to the MmPylRS/MmtRNA pair and show that transplanting mutations that reprogram the ncAA specificity of MmPylRS into the new PylRS reprograms its substrate specificity. Finally, we show that distinct PylRS/tRNA-derived pairs can function in the same cell, decode distinct codons and incorporate distinct ncAAs.