Background: A large study of intratumoral heterogeneity (ITH) in gastric cancer (GC) somatic mutations, a crucial consideration for effective druggability, has not been published. This study used targeted multi-regional sequencing (MSEQ) of gastric tumor DNA to examine cancer gene mutations, with the hypothesis of widespread ITH.Methods: MSEQ was carried out with a panel of >700 cancer-associated genes in 115 tumor biopsies from 32 patients. Analyses focused on identifying clonal and subclonal mutations in genes targetable with molecular therapies, comparing mutated genes to TCGA drivers, and exploring differences between this Latino-heavy population and the TCGA Latino-light study.Findings: Non-clonal mutations dominated, with relatively few clonal mutations. Only 33 of 54 known TCGA GC drivers harbored clonal mutations and several were never clonally mutated. Clonal mutations were found recurrently in potential new driver genes such as EYS, FAT4, PCDHA1, RAD50, and FSIP2. Only half of all tumors (16 of 32) harbored clonal mutations in druggable genes. The GS molecular subtype was more common in this patient population than in TCGA and was the only one where many patients (5 of 14) lacked any clonal mutations and the majority (10 of 14) lacked actionable clonal mutations discoverable with our panel. Mutation signatures revealed differing causes of clonal and non-clonal mutations, including tobacco carcinogens and deficiency of homologous recombination, mismatch repair, and POLE function.Interpretation: This first study to assess ITH in GC found extensive heterogeneity with rare clonal mutations, suggesting that single biopsies will often identify non-clonal mutations for drug targeting, and that existing GC drivers may frequently be non-clonally mutated. The results are important for improving molecular diagnostics by furthering the understanding of the genetic diversity and clonal architecture of these tumors.Funding Information: Funding sources for this study were: Universidad del Tolima, Colombia (Projects 160120516, 470115, 30113, 350113, 160114, 450110, 40218, 250120; contract 398-2017; grant 001-2019); MINCIENCIAS, Colombia (grants 850-2019, contract 940-2019); COLCIENCIAS, Colombia (grant 110565843382; contract 204 -2015; Graduate Studentships 647/2014 and 55/2016); L’OREAL-UNESCO-ICETEX-COLCIENCIAS, Colombia (project 3900917/2017); Instituto Mexicano del Seguro Social and Consejo Nacional de Ciencia y Tecnología, México (FIS/IMSS/PROT/PRIO/13/027); Consejo Nacional de Ciencia y Tecnología, México (Fronteras de la Ciencia 2015-01-773); The Auburn Community Endowed Chair in Basic Cancer Research, U.S; and the U.S. National Cancer Institute of the National Institutes of Health (grants R01CA223978, R21CA199631, U54CA233306 and P30CA093373). Declaration of Interests: None of the authors have conflicts to declare.Ethics Approval Statement: Research protocols used to recruit participants adhered to the Common Rule and were approved by Internal Review Boards at all participating institutions.