Recent advances in transcriptome sequencing have revealed that vertebrate genomes encode a huge variety of different RNA species that play fundamental roles in altering molecular mechanisms. Transfer RNAs (tRNAs) represent one of the most abundant classes of small noncoding RNAs. They act as crucial adapter molecules during protein synthesis by delivering amino acids into the ribosome to facilitate the decoding of messenger RNAs and are hence indispensable in every living organism. By using comparative genomics and deep sequencing technology, we will compare the evolutionary dynamics of tRNA genes between mammals and birds as well as within each vertebrate class to decipher the selective and mutational processes leading to tRNA gene evolution. Determining the number of tRNA genes, patterns of conserved and diverged synteny to tRNA genes, isoacceptor and isotype distribution will not only shed light into vertebrate genome organization and architecture but will also serve as a model for evolutionary processes contributing to human pathologies.