Background: ATP-binding cassette (ABC) transporters comprise a superfamily of genes encoding membrane proteins with nucleotide-binding domains (NBD). These transporters, including drug efflux across the blood–brain barrier (BBB), carry a variety of substrates through plasma membranes against substrate gradients, fueled by hydrolyzing ATP. The expression patterns/enrichment ofABCtransporter genes in brain microvessels compared to peripheral vessels and tissues are largely uncharacterized. Methods: In this study, the expression patterns ofABCtransporter genes in brain microvessels, peripheral tissues (lung, liver and spleen) and lung vessels were investigated using RNA-seq and WesTManalyses in three species: human, mouse and rat. Results: The study demonstrated thatABCdrug efflux transporter genes (includingABCB1,ABCG2,ABCC4andABCC5) were highly expressed in isolated brain microvessels in all three species studied; the expression ofABCB1,ABCG2,ABCC1,ABCC4andABCC5was generally higher in rodent brain microvessels compared to those of humans. In contrast,ABCC2andABCC3expression was low in brain microvessels, but high in rodent liver and lung vessels. Overall, mostABCtransporters (with the exception of drug efflux transporters) were enriched in peripheral tissues compared to brain microvessels in humans, while in rodent species, additionalABCtransporters were found to be enriched in brain microvessels. Conclusions: This study furthers the understanding of species similarities and differences in the expression patterns ofABCtransporter genes; this is important for translational studies in drug development. In particular, CNS drug delivery and toxicity may vary among species depending on their unique profiles ofABCtransporter expression in brain microvessels and BBB.