Background: High-throughput technologies have been increasingly used in research. Nevertheless, limited data are available on the feasibility and clinical value of these technologies in routine practice. Methods: All consecutive GI cancer patients (pts) who were tested with the 48-gene Truseq Amplicon Cancer Panel (Illumina) as part of routine practice at our Institution were identified from a prospectively maintained pathology database. Feasibility, results and impact on management decisions were analysed. Associations were tested with Fisher’s test. Results: From Apr 2014 to Jun 2019, 314 pts were tested. Sequencing was successful in 290 cases (92.4%; 234 colorectal (CRC), 21 gastro-esophageal, 17 bilio-pancreatic, 9 GIST, 4 appendix, 3 small intestine, 2 hepatocellular). In successful and failed cases, respectively, analyses were attempted on core biopsy (37.0% vs 66.7%), surgery (62.6% vs 16.7%), and fine needle aspiration (0.4% vs 12.4%) tumour samples (p < 0.001). Median turnaround time (TAT) was 12.5 days, reducing from 13 days in 2014 to 10 days in 2019. The majority of successfully tested pts (85.6%) had stage IV disease. TP53 was the most frequent mutation (45.9%), followed by APC (42.1%), KRAS (39.7%), PIK3CA (12.1%), SMAD4 (7.6%), BRAF (6.2%), and NRAS (5.5%). All other mutations were found in < 5% of cases. Excluding RAS/BRAF and KIT/PDGFR mutations, sequencing results impacted on the management of 4 (1.4%) pts. In 199 stage IV CRC pts, KRAS mutations were associated with lung (p = 0.020) and bone metastases (mts) (p = 0.008), NRAS mutations with liver mts (p = 0.021), BRAF mutations with peritoneal mts (p = 0.001), and APC mutations with lymph node mts (p = 0.006). Assuming a median of 25 samples per run, the estimated cost of NGS analysis per pt was €186. Conclusions: Low failure rate, short TAT and fair costs support feasibility of multi-gene NGS in routine practice. The clinical impact of this technology, however, is still limited but it could raise with the identification of new biologically relevant genetic alterations and the increased availability of novel therapeutics.