Posttranscriptional modifications of transfer RNAs (tRNAs) are critical for all core aspects of tRNA function, such as folding, stability, and decoding. Most tRNA modifications were discovered in the 1970s; however, the near-complete description of the genes required to introduce the full set of modifications in both yeast and Escherichia coli is very recent. This led to a new appreciation of the key roles of tRNA modifications and tRNA modification enzymes as checkpoints for tRNA integrity and for integrating translation with other cellular functions such as transcription, primary metabolism, and stress resistance. A global survey of tRNA modification enzymes shows that the functional constraints that drive the presence of modifications are often conserved, but the solutions used to fulfill these constraints differ among different kingdoms, organisms, and species.