Non-celiac wheat sensitivity (NCWS), also referred to as non-celiac gluten sensitivity, is a recently described disorder triggered by wheat/gluten ingestion. NCWS elicits a wide range of symptoms including diarrhoea, intestinal discomfort, and fatigue in analogy with other wheat/gluten-related disorders and celiac disease in particular. From the pathological standpoint, NCWS patients only have a slight increase of intraepithelial lymphocytes, while antibodies to tissue transglutaminase (tTG) and villous atrophy, otherwise diagnostic features of celiac disease, are absent. To date, the diagnosis of NCWS relies on symptoms and exclusion of confounding diseases, since biomarkers are not yet available. Here, the expression levels of selected miRNAs were examined in duodenal biopsies and peripheral blood leukocytes collected from newly diagnosed patients with NCWS and, as controls, from patients with celiac disease and gluten-independent gastrointestinal problems. We identified a few miRNAs whose expression is higher in the intestinal mucosa of patients affected by NCWS in comparison to control patients affect by gluten-independent dyspeptic symptoms (Helicobacter pylori-negative) and celiac disease. The present study provided the first evidence that NCWS patients have a characteristic miRNA expression patterns, such peculiarity could be exploited as a biomarker to the diagnosis of this disease.