Background: Colorectal cancer (CRC) is the third most common cancer worldwide, with metastatic disease accounting for 40 to 50% of newly diagnosed patients. EGFR monoclonal antibodies (Mab), cetuximab and panitumumab, are effective treatment for KRAS wild type CRC. Although mutations in KRAS predict resistance to EGFR Mab therapy, only 80% of CRC patients with KRAS wild-type (WT) status respond to treatment. This study is a retrospective evaluation of genomic alterations in the EGFR pathway such as alterations in KRAS, BRAF, NRAS, PIK3CA and PTEN that may predict lack of response to EGFR Mab therapy in CRC patients. Methods: A large database of 5,900 consecutive CRC patients was analyzed from 2011 onwards for demographics (sex, age, geography, site of tumor) and biomarkers that might correlate with response to EGFR Mab therapy. Comprehensive testing included gene sequencing for KRAS, BRAF, NRAS, PTEN, and PIK3CA (next-generation sequencing, Sanger, pyrosequencing) and immunohistochemistry for PTEN protein expression. Results: Analysis revealed the incidence of KRAS mutation of 42% which is consistent with published literature. Analysis of the KRAS WT cohort revealed a mutation rate of 13.8 % for BRAF, 6.8% for NRAS, and 40% for patients with either PTEN loss of expression/mutation or a PIK3CA mutation. Earlier studies support that mutations in NRAS, BRAF and activation of the PI3K pathway by PTEN/PIK3CA analysis result in lower response rates to EGFR Mab therapy in CRC. Furthermore, multiplex biomarker testing revealed the rare occurrence of concurrent mutations: 1/5,900 subject harboring a BRAF and NRAS mutations, 7/5,900 others with BRAF and PIK3CA mutations, and 8/5,900 subjects with NRAS and PIK3CA mutations. Conclusions: This is a comprehensive analysis of a large international cohort evaluating the prevalence of predictive molecular aberrations suspected of lack of response to EGFR Mab therapy in patients with WT KRAS. Prospective controlled studies are in progress to validate the role of BRAF, NRAS, PIK3CA, and PTEN in clinical management of CRC. It is imperative to further explore the molecular pathology of CRC beyond KRAS in patient selection for EGFR Mab therapy.