EPHrin receptors (EPH) are transmembrane receptor tyrosine kinases. Their extracellular domains bind specifically to ephrin A/B ligands, and this binding modulates the intracellular kinase activity. EPHs are key players in bidirectional intercellular signalling, controlling cell morphology, adhesion and migration. They are increasingly recognized as cancer drug targets. We analysed the binding of the patented Novartis inhibitor NVP-BHG712 (NVP) to EPHA2 and EPHB4. Unexpectedly, all tested commercially available NVP samples were regioisomers NVPiso with a single methyl group binding to an adjacent nitrogen atom compared to the patented compound. The two compounds of identical mass reveal different binding modes. Further, both, in vitro and in vivo experiments show that the isomers differ in their kinase affinity and selectivity.