Yeast mitochondrial DNA contains a genetic locus, called the tRNA synthesis locus, which codes for information necessary for mitochondrial tRNA biosynthesis. A 9S RNA molecule coded by this locus is thought to be the trans -acting element required for the removal of 5′ extensions from tRNA precursors. The DNA coding for this RNA maps to a region of mitochondrial DNA known to contain strain specific restriction site polymorphisms. Comparison of the tRNA synthesis locus in two such strains by sequence analysis demonstrates that the restriction enzyme polymorphisms are due to the deletion/insertion of a 50 base pair GC-rich element in the 5′ flanking sequence of the 9S RNA coding region. There are also several differences between the 9S RNA coding region of these two strains which do not interfere with the tRNA synthesis function.