The first description of Parvovirus B19 (B19V) infection in Manaus (AM) occurred in 1999, during the first dengue epidemic, when anti-IgM antibodies to B19V were detected in the serum of patients with a rash, but negative for dengue. However, molecular studies were not performed to provide timely serotyping results. In this study, we identified for the first time, using molecular methods, B19V in the sera of patients with acute febrile illness and negative for malaria and dengue, collected in the period 2001-2002 in Manaus, Amazonas. Samples from patients between 2 months and 67 years of age, negative for malaria and dengue, were tested for B19V by nested-PCR.  35 samples were positive; among these samples, two patients reported travel to another state and seven patients traveled to communities near Manaus and two patients lived in the countryside. Children and adults of both genders were infected, with symptoms similar to dengue. The presence of B19V during the first dengue epidemic in Amazonas with autochthonous cases shows that B19V has long been underreported due to the great clinical similarity between common viral diseases in the Amazonia, and hence it must be part of the differential diagnosis of etiological agents of acute febrile syndrome using molecular techniques that allow a fast and accurate diagnosis.