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Despite the recent advances in medicine, fever of unknown origin (FUO) remains a diagnostic and therapeutic challenge even to expert physicians. To increase the knowledge of FUO, we conducted a retrospective study to investigate the causes of FUO and the change of major causes of FUO during the past 26 years.The clinical data were retrospectively analyzed from 997 patients with FUO hospitalized at the Peking Union Medical College Hospital (PUMCH) between January 2004 and October 2010. Furthermore, the results were compared to that reported in previous studies of FUO in PUMCH since 1985.Of the 997 FUO cases, definite diagnosis was eventually achieved in 797 (79.9%) patients. The most common cause of FUO was infectious diseases (479 cases, 48.0%), with tuberculosis accounting for 45.3% (217/479) of the cases of infections. One hundred and sixty-eight (16.9%) patients were diagnosed with connective tissue diseases, with Still's disease and vasculitis accounted for 31.5% (53/168) and 24.4% (41/168) of this category, respectively. Neoplasms and miscellaneous causes were found in 7.9% (79/997) and 7.1% (71/997), respectively. However, no definite diagnosis had been made in the remaining 200 (20.1%) cases until they were discharged from the hospital.During different periods, infectious diseases, especially tuberculosis, were the leading etiology of FUO and the proportion of tuberculosis had no significant difference. While the frequency of neoplasms was descending, the proportion of lymphoma in neoplasm was ascending; the frequency of undiagnosed cases was increasing, but in most FUO cases the causes can be diagnosed eventually after careful analysis of clinical data.