Little information is available about Flavivirus infection in amerindian populations in western Venezuela. On this account the activity and seroprevalence of these viruses were determined and the hypothesis concerning the existence of a sylvatic cycle, conditioning the infection transmission of these viruses in indigenous populations, was studied. For this, blood samples from Yukpas (n=144) and Barí (n=110) communities were collected, 35 (Yukpas=25 and Barí=10) of which were processed for viral isolation followed by RT-PCR. The anti-Flavivirus IgG antibodies were determined by ELISA. The results did not show active Dengue cases and the seroprevalence of anti-Flavivirus IgG in the Yukpa population was significantly higher (p < 0.0001) than in the Barí population (43.1% vs. 6.4%). The present study has determined the presence of Flavivirus immunity in Yukpa and Barí populations. These results show a higher prevalence at the former than in the Barí population, which suggests circulation of Flavivirus, mainly in the Yukpa communities, being scarce and sporadic in Barí villages. However, in the indigenous populations studied, the causes or factors that determine the off set of Flavivirus infections in these zones could vary. The detected prevalence between both communities may be due to differences in the structure settlements and social habits. No evidences were found to support the presence of a sylvatic cycle in the Flavivirus transmission, specially of Dengue, in this population.