The epidermal growth factor receptor (EGFR) is a well-known oncogenic driver in lung and other cancers. In glioblastoma multiforme (GBM), the EGFR deletion variant III (EGFRvIII) is frequently found alongside EGFR amplification. Agents targeting the EGFR axis have shown limited clinical benefits in GBM and the role of EGFRvIII in GBM is poorly understood. To shed light on the role of EGFRvIII and its potential as a therapeutic target, we determined X-ray crystal structures of a monomeric EGFRvIII extracellular region (ECR). The EGFRvIII ECR resembles the unliganded conformation of EGFR, including the orientation of the C-terminal region of domain II. Domain II is mostly disordered, but the ECR structure is compact. We selected a nanobody with preferential binding to EGFRvIII relative to EGFR and structurally defined an epitope on domain IV that is occluded in the unliganded intact EGFR. These findings suggest new avenues for EGFRvIII targeting in GBM. Display omitted •The EGFRvIII extracellular region adopts the conformation seen in tethered EGFR•Domain III of EGFRvIII is accessible for ligand or antibody binding•Domain II is largely disordered, but the extracellular region is compact•A nanobody/VHH selective for EGFRvIII recognized an epitope on domain IV The EGFRvIII truncation variant is frequently found in glioblastoma multiforme but its role and potential as a therapeutic target remain controversial. Bagchi, Stayrook et al. determined the EGFRvIII extracellular region structure. They also describe a VHH/nanobody recognizing an unexpected epitope in domain IV that is occluded in unliganded intact EGFR.