Guaroa virus (GROV) was first isolated from humans in Colombia in 1959. Subsequent isolates of the virus have been recovered from febrile patients and mosquitoes in Brazil, Colombia, and Panama; however, association of the virus with human disease has been unclear. As part of a study on the etiology of febrile illnesses in Peru and Bolivia, 14 GROV strains were isolated from patients with febrile illnesses, and 3 additional cases were confirmed by IgM seroconversion. The prevalence rate of GROV antibodies among Iquitos residents was 13%; the highest rates were among persons with occupations such as woodcutters, fisherman, and oil-field workers. Genetic characterization of representative GROV isolates indicated that strains from Peru and Bolivia form a monophyletic group that can be distinguished from strains isolated earlier in Brazil and Colombia. This study confirms GROV as a cause of febrile illness in tropical regions of Central and South America. Copyright © 2010 by The American Society of Tropical Medicine and Hygiene.