© 2021 John Wiley & Sons LtdAims: The differential diagnosis of Fever of Unknown Origin (FUO) is still a major clinical challenge despite the advances in diagnostic procedures. In this multicentre study, we aimed to reveal FUO aetiology and factors influencing the final diagnosis of FUO in Turkey. Methods: A total of 214 patients with FUO between the years 2015 and 2019 from 13 tertiary training and research hospitals were retrospectively evaluated. Results: The etiologic distribution of FUO was infections (44.9%), malignancies (15.42%), autoimmune/inflammatory (11.68%) diseases, miscellaneous diseases (8.41%) and undiagnosed cases (19.62%). Brucellosis (10.25%), extrapulmonary tuberculosis (6.54%) and infective endocarditis (6.54%) were the most frequent three infective causes. Solid malignancies (7.1%) and lymphoma (5.6%), adult-onset still's disease (6.07%) and thyroiditis (5.14%) were other frequent diseases. The aetiological spectrum did not differ in elderly people (P <.05). Infections were less frequent in Western (34.62%) compared with Eastern regions of Turkey (60.71%) (P <.001, OR: 0.31, 95% Cl: 0.19 to 0.60). The ratio of undiagnosed aetiology was significantly higher in elderly people (p: 0.046, OR: 2.34, 95% Cl: 1.00 to 5.48) and significantly lower in Western Turkey (P:.004, OR: 3.07, 95% Cl: 1.39 to 6.71). Conclusions: Brucellosis, extrapulmonary tuberculosis and infective endocarditis remain to be the most frequent infective causes of FUO in Turkey. Solid tumours and lymphomas, AOSD and thyroiditis are the other common diseases. The aetiological spectrum did not differ in elderly people, on the other hand, infections were more common in Eastern Turkey. A considerable amount of aetiology remained undiagnosed despite the state-of-the-art technology in healthcare services.