Anti-epidermal growth factor receptor (EGFR) antibodies are effective treatments for metastatic colorectal cancer. Improved understanding of acquired resistance mechanisms may facilitate circulating tumor DNA (ctDNA) monitoring, anti-EGFR rechallenge, and combinatorial strategies to delay resistance. Patients with treatment-refractory metastatic colorectal cancer (n = 169) enrolled on the CO.26 trial had pre-anti-EGFR tissue whole-exome sequencing (WES) compared with baseline and week 8 ctDNA assessments with the GuardantOMNI assay. Acquired alterations were compared between patients with prior anti-EGFR therapy (n = 66) and those without. Anti-EGFR therapy occurred a median of 111 days before ctDNA assessment. ctDNA identified 12 genes with increased mutation frequency after anti-EGFR therapy, including ( = .0007), ( = .0017), ( = .0046), ( = .0086), ( = .018), ( = .018), ( = .048), and ( = .048). Acquired mutations appeared as multiple concurrent subclonal alterations, with most showing decay over time. Significant increases in copy-gain frequency were noted in 29 genes after anti-EGFR exposure, with notable alterations including ( < .0001), ( < .0001), ( < .0001), ( = .0002), ( = .0002), ( = .0006), ( = .004), and ( = .006). Copy gains appeared stable without decay 8 weeks later. There were 13 gene fusions noted among 11 patients, all but one of which was associated with prior anti-EGFR therapy. Polyclonal resistance was common with acquisition of ≥ 10 resistance related alterations noted in 21% of patients with previous anti-EGFR therapy compared with 5% in those without ( = .010). Although tumor mutation burden (TMB) did not differ pretreatment ( = .63), anti-EGFR exposure increased TMB ( = .028), whereas lack of anti-EGFR exposure resulted in declining TMB ( = .014). Paired tissue and ctDNA sequencing identified multiple novel mutations, copy gains, and fusions associated with anti-EGFR therapy that frequently co-occur as subclonal alterations in the same patient.