Background & Aims: Studies of non-celiac gluten or wheat sensitivity (NCGWS) have increased but there are no biomarkers of this disorder. We aimed to evaluate histologic features of colon and rectal tissues from patients with NCGWS. Methods: We performed a prospective study of 78 patients (66 female; mean age, 36.4 years) diagnosed with NCGWS by double-blind wheat challenge at 2 tertiary care centers in Italy, from January 2015 through September 2016. Data were also collected from 55 patients wither either celiac disease or self-reported NCGWS but negative results from the wheat-challenge test (non-NCGWS controls). Duodenal and rectal biopsies were collected and analyzed by immunohistochemistry to quantify intra-epithelial CD3 + T cells, lamina propria CD45 + cells, CD4 + and CD8 + T cells, mast cells, and eosinophils and to determine the presence and size of lymphoid nodules in patients with NCGWS vs patients with celiac disease or non-NCGWS controls. Results: Duodenal tissues from patients with NCGWS had significantly higher numbers of intra-epithelial CD3 + T cells, lamina propria CD45 + cells, and eosinophils than duodenal tissues from non-NCGWS controls. Duodenal tissues from patients with NCGWS and dyspepsia had a higher number of lamina propria eosinophils than patients with NCGWS without upper digestive tract symptoms. Rectal mucosa from patients with NCGWS had a larger number of enlarged lymphoid follicles, intra-epithelial CD3 + T cells, lamina propria CD45 + cells, and eosinophils than rectal mucosa from non-NCGWS controls. Duodenal and rectal mucosal tissues from patients with celiac disease had more immunocytes (CD45 + cells, CD3 + cells, and eosinophils) than tissues from patients with NCGWS or non-NCGWS controls. Conclusions: We identified markers of inflammation, including increased numbers of eosinophils, in duodenal and rectal mucosa from patients with NCGWS. NCGWS might therefore involve inflammation of the entire intestinal tract. Eosinophils could serve as a biomarker for NCGWS and be involved in its pathogenesis. Clinicaltrials.gov: NCT01762579.