Isoprenoids are a highly diverse and important group of natural compounds. The enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) catalyzes a key regulatory step in the non-mevalonate isoprenoid biosynthetic pathway in eubacteria and in plant plastids. For example, in Artemisia annua DXR participates in regulation of the biosynthesis of artemisinin, an important antimalarial drug. We performed phylogenetic analysis using DXR protein sequences from a model prokaryote, Escherichia coli, a picoplanktonic alga, Ostreococcus lucimarinus, and higher plants. The functional domain of DXR was conserved, allowing molecular evolutionary comparisons of both prokaryotic and eukaryotic sequences of DXR. Despite this conservation, for some plant species such as Campthoteca acuminata and Arabidopsis thaliana, phylogenetic relationships of their lineages were consistently violated. Our analysis revealed that plant DXR has an N-terminal transit domain that is likely bipartite, consisting of a chloroplast transit peptide (cTP) and a lumen transit peptide (lTP). Several features observed in the lTP suggest that, while DXR is targeted to the chloroplast, it is localized to the thylakoid lumen. These features include a twin arginine motif, a hydrophobic region, and a proline-rich region. The transit peptide also showed putative motifs for a 14-3-3 binding site with a chaperone phosphorylation site at Thr.