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Abstract Background On 28 March, 2016, the Ministry of Health received a report on three deaths from an unknown disease characterized by fever, jaundice, and hemorrhage which occurred within a one-month period in the same family in central Uganda. We started an investigation to determine its nature and scope, identify risk factors, and to recommend eventually control measures for future prevention. Methods We defined a probable case as onset of unexplained fever plus ≥1 of the following unexplained symptoms: jaundice, unexplained bleeding, or liver function abnormalities. A confirmed case was a probable case with IgM or PCR positivity for yellow fever. We reviewed medical records and conducted active community case-finding. In a case-control study, we compared risk factors between case-patients and asymptomatic control-persons, frequency-matched by age, sex, and village. We used multivariate conditional logistic regression to evaluate risk factors. We also conducted entomological studies and environmental assessments. Results From February to May, we identified 42 case-persons (35 probable and seven confirmed), of whom 14 (33%) died. The attack rate (AR) was 2.6/100,000 for all affected districts, and highest in Masaka District (AR = 6.0/100,000). Men (AR = 4.0/100,000) were more affected than women (AR = 1.1/100,000) (p = 0.00016). Persons aged 30–39 years (AR = 14/100,000) were the most affected. Only 32 case-patients and 128 controls were used in the case control study. Twenty three case-persons (72%) and 32 control-persons (25%) farmed in swampy areas (ORadj = 7.5; 95%CI = 2.3–24); 20 case-patients (63%) and 32 control-persons (25%) who farmed reported presence of monkeys in agriculture fields (ORadj = 3.1, 95%CI = 1.1–8.6); and 20 case-patients (63%) and 35 control-persons (27%) farmed in forest areas (ORadj = 3.2; 95%CI = 0.93–11). No study participants reported yellow fever vaccination. Sylvatic monkeys and Aedes mosquitoes were identified in the nearby forest areas. Conclusion This yellow fever outbreak was likely sylvatic and transmitted to a susceptible population probably by mosquito bites during farming in forest and swampy areas. A reactive vaccination campaign was conducted in the affected districts after the outbreak. We recommended introduction of yellow fever vaccine into the routine Uganda National Expanded Program on Immunization and enhanced yellow fever surveillance.