The primary function of tRNA genes is to provide the templates for the transcription of essential tRNA molecules. However, there is now evidence that these dispersed repetitive elements have the potential to mediate the spatial and functional organization of the genome and to drive genome change and evolution. Indeed, tRNA genes and related Pol III promoter elements can occupy distinct subnuclear positions and also provide barriers which functionally separate domains of chromatin. Furthermore, tRNA genes can also represent barriers to DNA replication fork progression and accordingly, tRNA genes can contribute to the formation of genomic fragile sites and have been implicated in genome evolution. Here we give insight into our current understanding of these "extra transcriptional" functions of tRNA genes and discuss how these functions may impact upon genome regulation and evolution.