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Background Southern China is at risk for arborvirus disease transmission, including Zika virus and dengue. Patients often present to clinical care with non-specific acute febrile illnesses (AFI). To better describe the etiology of AFI, we implemented a two-year AFI surveillance project at five sentinel hospitals in Yunnan and Guangdong Provinces. Methods Between June 2017 and August 2019, we enrolled patients between 2 and 65 years of age presenting at one sentinel hospital in Mengla County, Yunnan, and four in Jiangmen City, Guangdong, with symptoms of AFI (acute onset of fever ≥ 37.5°C within the past 7 days) without respiratory symptoms or diarrhea. Demographic, epidemiologic, and clinical information was obtained and entered into a web-based AFI surveillance database. A custom TaqMan Array card (TAC) was used to test patients’ whole blood specimens for 27 different pathogens using real-time polymerase chain reaction assays. Results During the two-year project period, 836 patients were enrolled; 443 patients from Mengla County and 393 patients from Jiangmen City. The median age was 33 years [range: 2–65], and most were hospitalized [641, 77%]. Of 796 patients with valid TAC results, 341 (43%) were positive for at least one of the 10 unique pathogens detected. This included 205 (26%) patients positive for dengue virus, 60 (8%) for Orientia tsutsugamushi, and 42 (5%) for Coxiella burnetii. Ten patients (1%) in Jiangmen City tested positive for malaria, 8 of whom reported recent travel outside of China. TAC results were negative for 455 (57%) patients. None of the patients had a positive TAC detection for Zika virus. Conclusions The project detected variability in the etiology of AFI in Southern China and highlighted the importance of differential diagnosis. Dengue, O. tsutsugamushi, and C. burnetii were the most frequently identified pathogens among enrolled AFI patients. As a non-notifiable disease, the frequent detection of C. burnetii is noteworthy and warrants additional investigation. The project provided a framework for routine surveillance for persons presenting with AFI.