Mutations in are considered to be the main drivers of acquired resistance to epidermal growth factor receptor (EGFR) blockade in patients with metastatic colorectal cancer (mCRC). However, the potential role of other genes downstream of the EGFR signaling pathway in conferring acquired resistance has not been extensively investigated. Using circulating tumor DNA (ctDNA) from patients with mCRC and with acquired cetuximab resistance, we developed a targeted amplicon ultra-deep sequencing method to screen for low-abundance somatic mutations in a panel of genes that encode components of the EGFR signaling pathway. Mutations with significantly increased variant frequencies upon disease progression were selected by using quartile analysis. The functional consequences of the identified mutations were validated in cultured cells. We analyzed 32 patients with acquired cetuximab resistance in a development cohort. Of them, seven (22%) carried five novel mutations, whereas eight (25%) carried previously reported mutations. Functional studies showed that novel mutations (all in exon 19; p.K944N, p.F930S, p.V955G, p.V955I, and p.K966E) promote cell viability in the presence of cetuximab. Only one novel mutation (p.K944N) was verified in one of the 27 patients with acquired resistance in a validation cohort, simultaneous and hotspot mutations were detected in two patients. Among the above 59 acquired resistance patients, those with or mutations detected in ctDNA showed a pronounced decrease in progression-free survival than patients with no mutation. The mutations may potentially contribute to acquired cetuximab resistance in patients with mCRC. .