The discriminator base N73 is a key identity element of tRNA. In eukaryotes, N73 is an "A" in cytoplasmic tRNA and a "C" in mitochondrial tRNA. We present evidence herein that yeast histidyl-tRNA synthetase (HisRS) recognizes both A73 and C73, but somewhat prefers A73 even within the context of mitochondrial tRNA. In contrast, humans possess two distinct yet closely related HisRS homologues, with one encoding the cytoplasmic form (with an extra N-terminal WHEP domain) and the other encoding its mitochondrial counterpart (with an extra N-terminal mitochondrial targeting signal). Despite these two isoforms sharing high sequence similarities (81% identity), they strongly preferred different discriminator bases (A73 or C73). Moreover, only the mitochondrial form recognized the anticodon as a strong identity element. Most intriguingly, swapping the discriminator base between the cytoplasmic and mitochondrial tRNA isoacceptors conveniently switched their enzyme preferences. Similarly, swapping seven residues in the active site between the two isoforms readily switched their N73 preferences. This study suggests that the human HisRS genes, while descending from a common ancestor with dual function for both types of tRNA, have acquired highly specialized tRNA recognition properties through evolution.