AbstractNucleotides of transfer RNAs (tRNAs) are highly modified, particularly at the anticodon. Bacterial tRNAs that read A‐ending codons are especially notable. The U34 nucleotide canonically present in these tRNAs is modified by a wide range of complex chemical constituents. An additional two A‐ending codons are not read by U34‐containing tRNAs but are accommodated by either inosine or lysidine at the wobble position (I34 or L34). The structural basis for many N34 modifications in both tRNA aminoacylation and ribosome decoding has been elucidated, and evolutionary conservation of modifying enzymes is also becoming clearer. Here we present a brief review of the structure, function, and conservation of wobble modifications in tRNAs that translate A‐ending codons. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1158–1166, 2019