Phosphotyrosine binding (PTB) domains of the adaptor proteins Doks (downstream of tyrosine kinases) play an important role in regulating signal transduction of cell-surface receptors in cell growth, proliferation and differentiation; however, ligand specificity of the Dok PTB domains has until now remained elusive. In this study, we have investigated the molecular basis of specific association between the Dok1 PTB domain and the tyrosine-phosphorylated EGFR. Using yeast two-hybrid and biochemical binding assays, we show that only the PTB domain from Dok1 but not Dok4 or Dok5 can selectively bind to two known tyrosine phosphorylation sites at Y1086 and Y1148 in EGFR. Our structure-based mutational analyses define the molecular determinants for the two distinct Dok1 PTB domain/EGFR interactions and provide the structural understanding of the specific interactions between EGFR and PTB domains in the divergent Dok homologues.