Summary Objectives This study aimed to describe the causes of acute undifferentiated fever (AUF) and to develop a robust definition of undiagnosed undifferentiated fever (UUDF). Methods This was a retrospective study of AUF over 3 years (2008–2011) in an Australian tertiary hospital. Request for laboratory investigation of one or more infectious agents was used as the search tool. Results A total of 340 patients with AUF, aged 15–65 years, were identified over the study period. A final diagnosis was made in 147 (43.2%) patients, dengue fever being the most frequent. The aetiology of fever was not determined in 193 (56.8%) patients. Elevations of C-reactive protein (CRP) and hepatic aminotransferase levels were common in these patients; two patients died. The characteristics of UUDF were fever for ≤21 days and failure to reach a diagnosis after clinical evaluation and specific laboratory investigations. Conclusion The high burden of UUDF argues for a better diagnostic approach to fever that is capable of identifying a broad range of infectious agents.