Mitochondrial gene order characters are a promising, under utilised source of phylogenetic data for addressing long standing problems in insect evolution. In this study we examined the utility of transfer RNA gene rearrangements to address the issue of high level relationships within the Order Lepidoptera. The ground plan gene order of Ile - Gln - Met - ND2 is found in early branching, non-ditrysian superfamilies and the sister order Trichoptera. The derived arrangement Met - Ile - Gln - ND2 is found in all ditrysian species examined and appears to be a molecular synapomorphy for Ditrysia. Additionally the region between Gln and ND2 is retained in all examined Ditrysia as a spacer region. The retention of this spacer for 150 million years (since upper Jurasic) suggests that it may function in genome signaling. The evolution of this tRNA region and the spacer will be discussed.