Key Findings: 

• Analysis of 823 small bowel tumor samples with DNA and RNA sequencing represents the largest molecular profile study to date of this rare cancer type. 
• Subsites of small bowel tumors were shown to present distinct molecular features, suggesting differential treatment opportunities. 
• Different subsites were also enriched for targetable alterations, including HER2 overexpression and ERBB2 amplification in duodenal; RSPO3 fusions and BRAF and ERBB2 (HER2) mutations in jejunal; and mutations in genes encoding DNA damage repair proteins in ileal small bowel adenocarcinomas. 
• Comparison with a large cohort of colorectal cancer samples revealed dramatically different molecular landscapes, tumor microenvironment characteristics, and clinical outcomes, justifying the recent addition of small bowel adenocarcinoma-specific guidelines to clinical practice and highlighting the need for molecular profiling to inform cancer therapy.