BACKGROUND: The overlap between functional dyspepsia (FD) and irritable bowel syndrome (IBS) is associated with more severe gastrointestinal (GI) symptoms and lower quality of life. However, the gender differences in FD-IBS overlap remain unclear. This study aimed to investigate the gender differences in patients with FD-IBS overlap. METHODS: Controls and cases were prospectively enrolled from July 2019 to June 2020 at nine tertiary referral centers. The patients underwent esophagogastroduodenoscopy, and their symptoms were evaluated using a questionnaire including GI symptom-related items and the Hospital Anxiety Depression Scale (HADS). FD and IBS were diagnosed according to the Rome IV criteria, and the clinical characteristics of the FD-IBS overlap group were compared with those of the FD-only or IBS-only group. RESULTS: Among 667 subjects (334 healthy controls, 168 with FD-only, 37 with IBS-only, 128 with FD-IBS overlap), the FD-IBS overlap group (19.2%) showed a higher rate of preference for dairy products, a higher rate of history of Helicobacter pylori eradication, and higher HADS scores than the non-overlap group (P < 0.05). In the FD-IBS overlap group, men complained of reflux symptom and loose/watery stools more than women (P < 0.05), whereas women showed more severe GI symptoms, especially epigastric pain/burning symptoms, and higher depression scores than men (all P < 0.05). CONCLUSIONS: FD-IBS overlap patients are associated with severe upper GI symptoms and depression compared to non-overlap patients. Moreover, women with FD-IBS overlap experience more severe GI and depression symptoms than men.