Platelet-derived growth factor receptor (PDGFR) β is a marker of stromal pericytes and fibroblasts and represents an interesting target for both diagnosis and therapy of solid tumors. A receptor-specific imaging agent would be a useful tool for further understanding the prognostic role of this receptor in vivo. Affibody molecules constitute a class of very small binding proteins that are highly suited for in vivo imaging applications and that can be selected to specifically recognize a desired target protein. Here we describe the isolation of PDGFRβ-specific Affibody molecules with subnanomolar affinity. First-generation Affibody molecules were generated from a large naive library using phage display selection. Subsequently, sequences from binders having a desired selectivity profile and competing with the natural ligand for binding were used in the design of an affinity maturation library, which was created using a single partially randomized oligonucleotide. From this second-generation library, Affibody molecules with a 10-fold improvement in affinity (Kd=0.4–0.5 nM) for human PDGFRβ and a 4-fold improvement in affinity (Kd=6–7 nM) for murine PDGFRβ were isolated and characterized. Complete reversible folding after heating to 90 °C, as demonstrated by circular dichroism analysis, supports tolerance to labeling conditions for molecular imaging. The binders were highly specific, as verified by dot blot showing staining reactivity only with human and murine PDGFRβ, but not with human PDGFRα, or a panel of control proteins including 16 abundant human serum proteins. The final binder recognized the native conformation of PDGFRβ expressed in murine NIH-3T3 fibroblasts and human AU565 cells, and inhibited ligand-induced receptor phosphorylation in PDGFRβ-transfected porcine aortic endothelial cells. The PDGFRβ-specific Affibody molecule also accumulated around tumoral blood vessels in a model of spontaneous insulinoma, confirming a potential for in vivo targeting.