Significance Aminoacyl-tRNA synthetases (aaRSs) establish the rules to express the universal genetic code. During aminoacylation, each of the 20 aaRSs associates 1 of 20 amino acids with a specific trinucleotide known as anticodon. Remarkably, for alanyl-tRNAs, the synthetase makes no contact with the anticodon. Instead, it uses a “second genetic code” by picking out a single G3:U70 base pair in the tRNA acceptor stem, which is close to the amino acid attachment site, but 76 Å away from the anticodon. Here, we show that, while in the three kingdoms of life, alanyl-tRNA synthetases use G3:U70 to identify alanyl-tRNAs, surprisingly, they use three different mechanisms to achieve this. We thus suggest that, in evolution, the genetic code had a powerful and persistent preference for associating G:U with alanine.