Comprehensive genomic profiling (CGP) testing by next-generation sequencing has been introduced into clinical practice as part of precision cancer medicine to select effective targeted therapies. However, whether CGP testing at the time of first-line chemotherapy could be clinically useful is not clear. We conducted this single-center, prospective, observational study to investigate the feasibility of CGP testing for chemotherapy-naïve patients with stage III/IV gastrointestinal cancer, rare cancer, and cancer of unknown primary, using the FoundationOne® companion diagnostic (F1CDx) assay. The primary outcome was the detection rate of at least one actionable/druggable cancer genomic alteration. Actionable/druggable cancer genomic alterations were determined by the F1CDx report. An institutional molecular tumor board determined the molecular-based recommended therapies. A total of 197 patients were enrolled from October 2018 to June 2019. CGP success rate was 76.6% (151 of 197 patients), and median turnaround time was 19 days (range: 10-329 days). Actionable and druggable cancer genomic alterations were reported in 145 (73.6%) and 124 (62.9%) patients, respectively. The highest detection rate of druggable genomic alterations in gastrointestinal cancers was 80% in colorectal cancer (48 of 60 patients). Molecular-based recommended therapies were determined in 46 patients (23.4%). CGP testing would be a useful tool for the identification of a potentially effective first-line chemotherapy.