Background: Peanut allergic individuals can exhibit clinically-relevant IgE cross-reactivity to tree nuts, particularly walnut. Vicilin-buried peptides (VBPs) in N-terminal vicilin leader sequences (LS) in peanut and tree nuts contribute to IgE cross-reactivity due to highly conserved and stable α-hairpinin fold with variable amino acid sequences. Cross-reactive IgE binding patterns to linear and conformational epitopes of peanut and walnut VBPs provides a model to understand clinically-relevant cross-reactivity. Methods: Sera IgE binding from oral food challenge positive peanut (PNA), walnut (WNA), and PN+WN (PWA) allergic subjects were assessed by microarray containing overlapping peptides of Ara h 1, Jug r 2, and LSs and by direct and competitive inhibition ELISA with VBPs from peanut (AH1.1), and walnut (JR2.1, JR2.2, JR2.3). Mixed model analysis was performed to investigate contribution of IgE binding to linear and conformational epitopes of VBPs to PNA, WNA, or PWA status. Results: All three intact and folded WN VBPs bound IgE at similar frequencies with individual sera preferentially recognizing one VBP over another; however, both weak and strong binding VBPs are competing with the same monoclonal IgE molecules within sera. AH1.1 was least recognized by WNA and most recognized by PNA and PWA. IgE binding patterns and specificities to VBPs were able to distinguish between WNA, PNA, or PWA groups. Conclusions: Cross-reactivity among VBPs is due to the binding of a monoclonal IgE in sera to α-hairpinin structures. IgE binding to the linear and conformational epitopes of Ara h 1 and Jug r 2 VBPs could be beneficial in discerning PNA and WNA.