Epidermal growth factor receptor’s (EGFR) upregulation is associated with increased proliferation and drug resistance to targeted therapies in almost all cancers of epithelial origin notably in colorectal cancer. Matuzumab is a monoclonal antibody (mAb) that binds to a unique epitope of domain III of EGFR with high affinity. Nimotuzumab is an anti-EGFR therapeutic mAb with favorable size effects compared with other approved anti-EGFR mAbs. The objective of this work is to develop 89Zr-matuzumab as a PET probe that allows for monitoring of response to treatment using other anti-EGFR antibodies that may bind to different epitopes. To accomplished this, we have conjugated matuzumab to deferoxamine (DFO), and radiolabeled with 89Zr. Quality control for this conjugate was performed using size-exclusion HPLC, bioanalyzer, and flow cytometry in EGFR positive colorectal cancer cell line DLD-1. The flow cytometry analysis showed that the binding of matuzumab-DFO (KD = 6.15 nM) was not significantly affected by the conjugation (vs matuzumab KD = 5.85 nM). Radioligand binding assay of 89Zr-matuzumab confirmed the binding of the radiolabeled 89Zr-DFO conjugate to EGFR positive DLD-1 cells (KD = 3.12 nM; Bmax = 22341). We also found out from competitive radioligand binding assay that matuzumab and nimotuzumab bind to different epitopes of EGFR. MicroPET/CT imaging and biodistribution of mice bearing two different EGFR positive colorectal cancer xenografts (HT29 and DLD-1) using 89Zr-matuzumab showed persistently high uptake in both xenografts. In vivo pre-blocking experiments with matuzumab and nimotuzumab further, confirm the epitope specificity of the two antibodies