Adenocarcinomas of the esophagus (EAC), stomach [gastric adenocarcinoma (GAC)], and colorectal carcinoma (CRC) frequently show similar morphology because upper gastrointestinal tumors (GITs) usually evolve from pathologies involving intestinal metaplasia. Upper and lower GIT may also show overlapping immunophenotypes when using the traditional CK7, CK20, and CDX2 panel, which in patients presenting with metastatic disease of unknown origin may lead to misdirected diagnostic workup and/or therapy. We compared the phenotype of upper and lower GIT using an expanded immunohistochemical panel that included the traditional and newer gastrointestinal markers: SATB2, DcR3, MUC5AC, and MUC6. The panel was applied to resection specimens from 40 CRC, 40 GAC, and 40 EAC. A panel using SATB2, CK7, and CDX2 provided the best discriminating power for separating upper from lower GIT and was applied to 101 biopsies including 17 EAC, 17 GAC, 19 CRC, 18 pancreatic adenocarcinomas, 15 cholangiocarcinomas, and 15 lung adenocarcinomas. The phenotype CK7/CDX2/SATB2 was moderately sensitive and highly specific of upper GIT, the phenotype CK7/CDX2/SATB2 was highly sensitive and specific for lower GIT, the phenotypes CK7/CDX2/SATB2 and CK7/CDX2/SATB2 favored pancreatobiliary or lung primaries. Less frequent phenotypes showed substantial overlap. Although strong diffuse expression of SATB2 was characteristic of CRC, weak and/or focal expression was present in one third or more of upper gastrointestinal, cholangiocarcinomas, and lung adenocarcinomas. DcR3, MUC5AC, and MUC6 improved specificity, but showed poor sensitivity, suggesting they should be used as second tier markers.