PURPOSE: The utility of capturing heterogeneity by circulating tumor DNA (ctDNA) genotyping combined with tissue analysis or applying it in a sequential manner remains uncertain. METHODS: We assessed the clinical value of ctDNA genotyping using data from 2,187 patients with advanced solid tumors enrolled in SCRUM-Japan MONSTAR-SCREEN-1, a nationwide cancer genome screening project, which examined ctDNA from longitudinally collected blood samples and tumor tissue samples (UMIN 000036749). RESULTS: Among 667 patients with both baseline ctDNA and tissue genotyping results, 51 (7.6%) had actionable biomarkers identified exclusively through ctDNA genotyping. The most frequent targets of genotype-matched therapy guided by solely ctDNA were immune checkpoint, estrogen receptor, and poly(ADP-ribose) polymerase (PARP). Comparison of objective response rates (ORRs) and progression-free survival (PFS) between patients treated based on tissue versus ctDNA alone showed no significant difference, with ORRs of 34.0% versus 23.1% (P = .54) and a median PFS of 11.5 versus 13.8 months (hazard ratio [HR], 1.4 [95% CI, 0.72 to 2.80]), respectively. Among 924 patients undergoing sequential ctDNA genotyping, the detection of actionable biomarkers increased from 63.2% to 72.5% following subsequent ctDNA. Targets for genotype-matched therapy guided by subsequent ctDNA alone commonly included PARP, immune checkpoint, and BRAF. The ORR was 23.2% and 26.7% (P = .75), and the median PFS was 5.2 and. 3.7 months (HR, 1.5 [95% CI, 0.79 to 2.80]) for genotype-matched therapy based on initial versus subsequent ctDNA alone, respectively. CONCLUSION: Combining ctDNA with tissue analysis, followed by sequential ctDNA assessments, effectively enhances the identification of actionable biomarkers. This strategy facilitates clinically beneficial, genetically informed therapies, underscoring its significant value in precision oncology.