In this study, we evaluated whether the presence of genetic alterations detected by next generation sequencing may define outcome in a prognostically-selected and histology-restricted population of resected gastric cancer (RGC). Intestinal type RGC samples from 34 patients, including 21 best and 13 worst prognostic performers, were studied. Mutations in 50 cancer-associated genes were evaluated. A significant difference between good and poor prognosis was found according to clinico-pathologic factors. The most commonly mutated genes in the whole population were PIK3CA (29.4%), KRAS (26.5%), TP53 (26.5%) MET (8.8%), SMAD4 (8.8%) and STK11 (8.8%). Multiple gene mutations were found in 14/21 (67%) patients with good prognosis, and 3/13 (23%) in the poor prognosis group. A single gene alteration was found in 5/21 (24%) good and 6/13 (46%) poor prognosis patients. No mutation was found in 2/21 (9.5%) and 4/13 (31%) of these groups, respectively. In the overall series, ß-catenin expression was the highest (82.4%), followed by E-Cadherin (76.5%) and FHIT (52.9%). The good prognosis group was characterized by a high mutation rate and microsatellite instability. Our proof-of-principle study demonstrates the feasibility of a molecular profiling approach with the aim to identify potentially druggable pathways and drive the development of customized therapies for RGC.