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Background Genomic analysis of circulating tumor cells (CTCs) could provide a unique and accessible representation of tumor diversity but remains hindered by technical challenges associated with CTC rarity and heterogeneity. Objective To evaluate CTCs as surrogate samples for genomic analyses in metastatic castration-resistant prostate cancer (mCRPC). Design, setting, and participants Three isolation strategies (filter laser-capture microdissection, self-seeding microwell chips, and fluorescence-activated cell sorting) were developed to capture CTCs with various epithelial and mesenchymal phenotypes and isolate them at the single-cell level. Whole-genome amplification (WGA) and WGA quality control were performed on 179 CTC samples, matched metastasis biopsies, and negative controls from 11 patients. All patients but one were pretreated with enzalutamide or abiraterone. Whole-exome sequencing (WES) of 34 CTC samples, metastasis biopsies, and negative controls were performed for seven patients. Outcome measurements and statistical analysis WES of CTCs was rigorously qualified in terms of percentage coverage at 10× depth, allelic dropout, and uncovered regions. Shared somatic mutations between CTCs and matched metastasis biopsies were identified. A customized approach based on determination of mutation rates for CTC samples was developed for identification of CTC-exclusive mutations. Results and limitations Shared mutations were mostly detected in epithelial CTCs and were recurrent. For two patients for whom a deeper analysis was performed, a few CTCs were sufficient to represent half to one-third of the mutations in the matched metastasis biopsy. CTC-exclusive mutations were identified in both epithelial and nonepithelial CTCs and affected cytoskeleton, invasion, DNA repair, and cancer-driver genes. Some 41% of CTC-exclusive mutations had a predicted deleterious impact on protein function. Phylogenic relationships between CTCs with distinct phenotypes were evidenced. Conclusions CTCs can provide unique insight into metastasis mutational diversity and reveal undiagnosed genomic aberrations in matched metastasis biopsies. Patient summary Our results demonstrate the clinical potential of circulating tumor cells to provide insight into metastatic events that could be critical to target using precision medicine.