Transfer RNAs are conserved RNAs that are essential for translation in all forms of life and are present in multiple variants to allow interpretation of the entire genetic code. Transfer RNA genes in eukaryotes exist in multiple identical and non-identical copies across the genome to contribute to the tRNA repertoire of a cell. While the same tRNAs are conserved across species, their number and conservation vary from genome to genome. The evolution of these tRNA genes, especially in mammals, is poorly understood. To understand what guides tRNA evolution, we examined the tRNA gene complement from 17 closely related mouse strain genomes. Our analysis revealed that although most tRNAs are conserved, a subset shows copy number variation, sequence divergence, and even strain-specific gains or losses. We also identified a group of transcribed single-copy tRNAs that are conserved across multiple mouse strains and other mammals, suggesting specialized or lineage-specific functions. Together, these findings provide new insights into the evolutionary dynamics of the mammalian tRNA repertoire.