Background: Cancer of unknown primary (CUP) with a “gastrointestinal (GI) profile” appears to be a clinically distinct subset, based on immunophenotyping, specifically staining with cytokeratins 20 (CK20) and 7 (CK7), a type I and II keratin, respectively and caudal type homeobox 2 (CDX2) protein, a transcription factor expressed in nuclei of intestinal epithelial cells. However, only a limited clinicomolecular account of this entity exists. Comprehensive profiling is needed to substantially impact personalized therapeutics and improve prognosis. Methods: We identified 401 pts evaluated at MD Anderson Cancer Center. Pts were classified into 3 cohorts based on immunohistochemistry: lower GI profile CUP (LGI-CUP), other-GI profile CUP (OGI-CUP), or non-GI CUP (NGI-CUP). A control group of known lower GI primary cancers was derived from MSK-IMPACT data (cBioPortal, 1075 patients, LGI-KP). Clinical and pathological data including molecular profiling, therapy and survival were logged. Fisher-exact test was used. Kaplan-Meier method was used to estimate overall survival (OS) and compared with log-rank test. Results: Among 748 pts, 401 (53.6%) had adequate immunostaining for analysis. Of these, 72 (18.0%), 226 (56.4%) and 103 (25.7%) were classified as LGI-CUP, OGI-CUP, and NGI-CUP, respectively. LGI-CUP were enriched for adenocarcinoma (88% v 86% v 35%, P < 0.0001) and good risk Culine score (59% v 51% v 37%, P = 0.02), compared to OGI-CUP and NGI-CUP, respectively. While NGI-CUP had greater proportions of poorly differentiated histology (91% v 55% vs 66%, P < 0.0001) and bone metastases (30% vs 7% v 24%, P = 0.0001) compared to LGI-CUP and OGI-CUP, respectively. Comparison of key mutations is shown in table. Median OS of LGI-CUP (14.6 months [95%CI: 12.0 – 17.2]) was similar to OGI-CUP (14.3 months [95%CI: 10.7 – 18.0]) and NGI-CUP (16.2 months [95%CI: 12.4 – 20.0]), P = 0.75, but significantly lower than LGI-KP cohort (28.6 months, [95%CI 24.4 – 32.7], p < 0.001). LGI-CUP was treated preferentially with fluoropyrimidine-based GI regimen [5-FU/capecitabine +/- oxaliplatin or irinotecan] (77% vs 43% v 5%) compared to OGI-CUP and NGI-CUP, respectively. However, median OS for LGI-CUP treated with 5-FU-based regimens was higher (16.0, 95%CI 13.0 – 19.0) than those treated with non-5-FU-based treatments (10.9, 95%CI 5.6 – 16.2), P = 0.002. Conclusions: LGI-CUP appears to be a molecularly distinct entity from and NGI-CUP, as well as lower GI CUP from known entities. LGI-CUP patients treated with 5-FU-based regimens appear to have improved survival.