Significant differences exist between the vascular endothelia of different organs, such as cell size, shape, mural cell coverage, and capillary permeability. To determine the epigenetic and transcriptional basis of this heterogeneity, we profiled accessible chromatin, as well as gene expression, across seven different organs, at three distinct time points, during murine development. After identifying both common, and organ-specific DNA motif usage and transcriptional signatures, we then focused our studies on the central nervous system (CNS) endothelium. Using single cell RNA-seq and informatic analyses, we identified key gene regulatory networks governing brain blood vessel maturation. Critically, these unique regulatory regions and gene expression signatures are evolutionarily conserved in humans. Collectively, this work provides a valuable lexicon for identifying the transcriptional regulators governing organ-specific endothelial specialization.