Patterns of substitution rates footprint functional constraints and highlight potential adaptive evolutionary change in macromolecules. The vast majority of molecular evolutionary studies have been on proteins. Even though tRNAs were the first RNAs to be sequenced and structurally solved, substitution rate analysis of tRNAs has not yet been published. In this dissertation, we advance the knowledge of tRNA evolution in eukaryotes, using sequence data from the twelve species of Drosophila. First I introduce background concepts covering the sequence, structure and function of tRNA, as well as describe early work studying the evolution of this molecule. In the second chapter, I describe divergence rates for tRNA sites, structures, and alloacceptor classes. I also discuss the evolution of the tRNA-protein interaction network in Drosophila with evidence of changes in “Class Informative Features” (CIFs) between species and clades of flies. In the third chapter, I discuss how work in site divergence rates lead us to investigate the effects of sequence mutations on one primary ion binding pocket through molecular dynamic simulations. Finally, I discuss some preliminary results using yeast sequence data to examine whether results from flies is specific to this group of species, or whether our results are generalizable to other eukaryotes. I also discuss the preliminary work using Drosophila melanogaster population sequences to ascertain the selective pressures acting upon tRNA sequences.