Overexpression of EGFR is a negative prognostic factor in head and neck squamous cell carcinoma (HNSCC). Patients with HNSCC who respond to EGFR-targeted tyrosine kinase inhibitors (TKIs) eventually develop acquired resistance. Strategies to identify HNSCC patients likely to benefit from EGFR-targeted therapies, together with biomarkers of treatment response, would have clinical value. Functional MRI and F-FDG PET were used to visualize and quantify imaging biomarkers associated with drug response within size-matched EGFR TKI-resistant CAL 27 (CAL ) and sensitive (CAL ) HNSCC xenografts , and pathological correlates sought. Intrinsic susceptibility, oxygen-enhanced and dynamic contrast-enhanced MRI revealed significantly slower baseline , lower hyperoxia-induced and volume transfer constant K in the CAL tumors which were associated with significantly lower Hoechst 33342 uptake and greater pimonidazole-adduct formation. There was no difference in oxygen-induced ΔR or water diffusivity between the CAL and CAL xenografts. PET revealed significantly higher relative uptake of F-FDG in the CAL cohort, which was associated with significantly greater Glut-1 expression. CAL xenografts established from HNSCC cells resistant to EGFR TKIs are more hypoxic, poorly perfused and glycolytic than sensitive CAL tumors. MRI combined with PET can be used to non-invasively assess HNSCC response/resistance to EGFR inhibition.