AbstractEmerging new mutations after treatment can provide clues to acquired resistant mechanisms. Circulating tumor DNA (ctDNA) sequencing has enabled noninvasive repeated tumor mutational profiling. We aimed to investigate newly emerging mutations in ctDNA after disease progression in metastatic colorectal cancer (mCRC). Blood samples were prospectively collected from mCRC patients receiving palliative chemotherapy before treatment and at radiological evaluations. ctDNA from pretreatment and progressive disease (PD) samples were sequenced with a next‐generation sequencing panel targeting 106 genes. A total of 712 samples from 326 patients were analyzed, and 381 pretreatment and PD pairs (163 first‐line, 85 second‐line and 133 later‐line [≥third‐line]) were compared. New mutations in PD samples (mean 2.75 mutations/sample) were observed in 49.6% (189/381) of treatments. ctDNA samples from later‐line had more baseline mutations (P = .002) and were more likely to have new PD mutations (adjusted odds ratio [OR] 2.27, 95% confidence interval [CI]: 1.40‐3.69) compared to first‐line. RAS/BRAF wild‐type tumors were more likely to develop PD mutations (adjusted OR 1.87, 95% CI: 1.22‐2.87), independent of cetuximab treatment. The majority of new PD mutations (68.5%) were minor clones, suggesting an increasing clonal heterogeneity after treatment. Pathways involved by PD mutations differed by the treatment received: MAPK cascade (Gene Ontology [GO]: 0000165) in cetuximab and regulation of kinase activity (GO: 0043549) in regorafenib. The number of mutations revealed by ctDNA sequencing increased during disease progression in mCRC. Clonal heterogeneity increased after chemotherapy progression, and pathways involved were affected by chemotherapy regimens.