Background: Small-bowel adenocarcinoma (SBA) is a rare malignancy with limited knowledge of the molecular mechanisms, or clinical evidence-based guidelines for therapy. We conducted a comprehensive analysis of biomarkers with therapeutic relevance for SBA. Methods: We examined the biomarker profiles of 266 SBA cases. Multiplatform biomarker panel included a combination of sequencing (Sanger, NGS), protein expression (IHC) and gene amplification (CISH /FISH). Results: Several biomarkers have been identified by IHC evaluation in ~250 patients: high TOP2A (74%) and low TUBB3 (81%), low ERCC1 (71%), low RRM1 (69%) and low TS (63%)- favorable for chemotherapy drugs such as doxorubicin, taxanes, oxaliplatin, gemcitabine and capecitabine, respectively. A minority of cases were found to express other favorable chemotherapy response biomarkers: low MGMT (alkylating agents, 30%), high SPARC (nab-paclitaxel, 36%) and high TOPO1 (irinotecan, 39%). SBA demonstrated a high prevalence of multidrug resistance gene expression: MRP1 and BCRP (83%) and PGP (51%). Unexpectedly, a number of cases showed genomic alterations associated with activated oncogenic signaling: EGFR (13% or 12/96), TOP2A (12% or 4/33), HER2 (7% or 9/130) and cMET (1.4% or 1/71). Two patients demonstrated co-amplification of TOP2A and HER2, suggesting these patients may benefit from anthracyclines. Evaluation of mutational hotspots demonstrated a high incidence of oncogenic/tumor suppressor mutations in 60% (47/78) of patients. We discovered that mutations in TP53 (51%) and KRAS (46%) were most common. Other genes with notable alterations included: APC (22%), SMAD4 (20%), BRAF (9%), CTNNB1 (7%) and PTEN (5%). Recent data suggest favorable responses to cetuximab in SBA; these results and high frequency of KRAS mutations in our series indicate that KRAS testing may be recommended in SBA. Conclusions: Our data demonstrate the potential utility of a wide range of traditional chemotherapies for SBA, targeted therapies utilized for other cancer types as well as therapies under clinical investigation. SBA exhibits biologically similar tumorigenesis as large-bowel adenocarcinoma, therefore similar treatment guidelines and biomarker testing strategies should be considered.